Objective To study the molecular epidemiology of babesiosis in cases of fever with thrombocytopenia in Xinyang City and provide basis for the control of babesiosis. Methods Blood samples and epidemiological information were collected from 600 patients who had fever and thrombocytopenia in Xinyang City in 2012. The DNA was extracted from the blood samples and Babesia 18S rRNA was amplified by nested-PCR with two sets of primers Bab5/Bab8 + Bab6/Bab7 and RIB-19/RIB-20 + BAB-rumF/BAB-rumR. The PCR products were sequenced and aligned with Babesia mocroti sequences in genome database. The PCR positive cases were further analyzed for their epidemiological characteristics, history of tick bite and bunyavirus infection. Results Totally 600 blood samples were collected from patients who had fever and thrombocytopenia, 59 (9.8%) of them were PCR positive with a fragment of 430 bp amplified by both sets of primers. The amplified 18S rRNA sequences shared more than 97% identity to LC314655.1 of B. mocroti. Among the 59 positive cases, 51 (86.4%) were from Xinyang City, 16 from Shangcheng County (27.1%) and 12 from Guangshan County (20.3%). The fever onset mostly happened in summer including 14 in May (23.7%), 23 in June (39.0%) and 10 in July (17.0%). Most of cases were middle-aged or elderly and 48 cases (81.4%) were over 40 years old. Among the 59 positive cases 14 (23.7%) had history to be bitten by ticks and 31 (52.5%) were positive for bunyavirus. Conclusion The morbidity of babesiosis is high in the cases with fever and thrombocytopenia in Xinyang City, Henan Province. People with middle-age and elderly are the most vulnerable population to be infected by Babesia during summer.