Objective To analyze and compare the biological characteristics and morphologies at different developmental stages beween Hyalomma asiaticum and H. anatolicum isolates from Xinjiang geographic regions. Methods Hard ticks were captured from Qitai and Turpan, the tick endemikc areas of Xinjiang, in April to May, 2019. Species of the ticks were identified based on morphology. The tick genomic DNA was extracted and the conserved fragment of the mitochondrial cytochrome oxidase subunit 1(CO1) gene was amplified by PCR. The target gene was cloned using pEASY-T1 vector system, single bacteria colony was selected for sterile expansion culture and the cultured fluid was undergone sequencing. The homology of CO1 gene sequence with other sequences was aligned using NCBI Blast online analysis software, and phylogenetic tree was generated. Twelve New Zealand white rabbits were divided into 2 groups with 6 rabbits in each group, H. asiaticum and H. anatolicum at different developmental stages (200 larvae, 150 nymphs, or 50 adults) were fed with blood from two rabbits. H. asiaticum and H. anatolicum ticks were placed in equipment enclosing rabbit ears to feed on blood. Ticks were checked daily until repletion. The recovered ticks were collected and stored under controlled experimental conditions. The biological characteristics of H. asiaticum and H. anatolicum was observed systematically at various developmental stages of their life cycles. Results A total of 317 H. asiaticum and 291 H. anatolicum ticks were collected in Qitai and Turpan, respectively. The results of phylogenetic analysis based on the CO1 gene show that H. asiaticum from Qitai, Xinjiang (MW217459), Inner Mongolia (JX051135), Kazakhstan (KU364324), Wuwei, Gansu (MK292004) and Yuli, Xinjiang (KF527441) belong to the same cluster; the sequence of H. anatolicum from Turpan, Xinjiang (MW221948), Kazakhstan (MN853167), Gansu (JQ737067) and other Xinjiang strains (MN268557, KF583576) were categorized into another cluster. There was significant difference in the genetic relationship between H. asiaticum-Qitai strain and H. anatolicum-Turpan strain. The average life cycles of H. asiaticum and H. anatolicum were 131.5 and 112.5 days, respectively. At the egg stage, the oviposition and egg’s incubation of H. asiaticum (27.5 days and 31.5 days) were longer than H. anatolicum (12.0 days and 20.5 days). At the larva stage, H. asiaticum and H. anatolicum need 5.5/18.5 days and 9/18.5 days to feed/molt, respectively. At the nymph stage, H. asiaticum and H. anatolicum need 10.5/19 days and 4.5/30 days to feed/molt, respectively. H. asiaticum and H. anatolicum needed 8.5/10.5 days and 13.0/5.0 days to feed/preoviposition. The morphological comparison showed that the appear time of “cicatricle” (later development to anus) in H. asiaticum (day 19) is later than H. anatolicum (day 14) in the process of egg development, but there were no significant difference between H. asiaticum and H. anatolicum at larvae and nymphs stages; the differences among adult ticks were mainly reflected in neck furrow (the male H. asiaticum has long and deep, extending backward beyond the middle of scutum, while that of the male H. anatolicum was short and shallow), paraproct and hypopygium (male H. asiaticum paraproct is narrow and long, and the anterior tip is narrow and the posterior tip is blunt and round, and the inner edge convex is wide and triangular, the hypopygium is slightly smaller and papillary; in male H. anatolicum, the paraproct is slightly wide in the middle, narrow in the front, slightly round and blunt in the back, and short in the inner edge, hypopygium is vague), spiracular plate (in male H. asiaticum, it is in the shape like a curved-neck-bottle, the front end is oval, the dorsum process is slightly narrow and long; in male H. anatolicum: it is in the shape like a spoon, dorsal process slightly wider and longer, end at the edge of the backplane. In female H. asiaticum: it is long-elliptic, slightly wider and longer dorsum process, at right angles to the back, terminal to the edge of the backplane. In female H. anatolicum: it is long rectangular, dorsum slightly wider and longer, bent forward nearly at right angles), coxa (in female H. asiaticum, the outer spur of the coxaⅠ is slightly longer than the inner spur. The outer spur is short and coarse, tapering in sequence. In female H. anatolicum: the outer spur of coxaⅠis larger than the inner spur. The outer spur of coxa Ⅱ~Ⅳ are short without the inner spur), and the colour banding of podomere (in male H. asiaticum it is clearly visible; in male H. anatolicum it is vague), among which the key identification character for male ticks were paraproct, hypopygium, and spiracular plate; the main identification character for female ticks were podomere and spiracular plate. Conclusion The biological characteristics of Xinjiang tick isolates H. asiaticum and H. anatolicum vary in life cycle, morphology, and molecular biology.