Objective To investigate the prevalence and genetic characteristics of common Apicomplexan protozoan in ticks collected from Chongming, Shanghai, so as to provide insights into management of tick-borne diseases in Chongming area. Methods Free-living ticks were collected using the flagging method in Chongming, Shanghai, from July to September 2023. Following morphological identification, ticks were pooled according to species and developmental stage (one adult per pool, five nymphs per pool, or ten larvae per pool), and genomic DNA was extracted from each pool. Tick species were molecularly identified by PCR amplification of the cytochrome c oxidase subunit 1 (cox1) gene and 16S rDNA gene. The short fragment of the Babesia/Theileria 18S rRNA gene (approximately 400 bp in length) was amplified using nested PCR assay, and positive samples were further subjected to amplification of the long fragment of the 18S rRNA gene (approximately 1 600 bp in length), and positive nested PCR products were sequenced, followed by sequence alignment with BLAST. Genetic distances were calculated among different samples using the software MEGA 11 and phylogenetic trees were created. Differences in the detection of protozoan were tested for statistical significance with chi-square test. Results A total of 622 free-living ticks were collected, including 255 Haemaphysalis longicornis and 367 H. flava. H. longicornis ticks were divided into 83 pools (55 adult pools, 16 nymph pools and 12 larval pools), and H. flava were divided into 43 pools (2 adult pools, 9 nymph pools and 32 larval pools). Nested PCR amplification of the short fragment of the 18S rRNA gene yielded 17 positive pools, including 16 H. longicornis pools (9 adult pools and 7 nymph pools) and one H. flava pool (adult pool). and the long fragment of the 18S rRNA gene was successfully amplified from all 17 positive pools, with sequences deposited under accession numbers of PX453257 to PX453273. BLAST alignments revealed that one gene sequence showed 99.52% identity with the Babesia 18S rRNA gene sequence (MK930513), 7 gene sequences showed 91.53% to 99.94% identity with the 18S rRNA gene sequences of the novel protozoan Colpodella, and 9 gene sequences showed 95.66% to 99.36% identity with the Colpoda 18S rRNA gene sequences. Genetic distance analysis indicated high intraspecific sequence conservation, with genetic distances ranging from 0.003 to 0.147 among 7 Colpodella gene sequences and from 0.009 to 0.060 among 9 Colpoda gene sequences, and the genetic distance between Colpodella and Colpoda ranged from 0.124 to 0.221, indicating substantial interspecific divergence. Phylogenetic analysis revealed that Colpoda and Colpodella were clustered into a large clade, and Babesia and Theileria were clustered into another large clade. The detection rates of Babesia, Colpodella, Colpoda, and protozoan were 1.20% (1/83), 8.43% (7/83), 9.64% (8/83), and 19.28% (16/83) in H. longicornis, and 0 (0/43), 0 (0/43), 2.33% (1/43), and 2.33% (1/43) in H. flava, respectively. The overall detection of protozoan was significantly higher in H. longicornis than in H. flava (χ² = 6.29, P < 0.05). Conclusion There were Babesia, Colpoda, and novel protozoan Colpodella infections in free-living ticks collected from Chongming area, indicating a potential risk of tick-borne diseases.