Objective To investigate and analyze the habitat and host distribution characteristics of tick species in Nanjing city to provide a basis for the prevention and control of ticks and tick-borne diseases.

Methods From April to September 2023, free-living and parasitic ticks were collected in Nanjing city by drag-flag method and body surface examination, respectively. The ticks were identified by morphological classification and molecular biological identification based on 16S rRNA gene.

Results A total of 1 523 ticks were collected, including 993 Haemaphysalis longicornis (65.20% of all ticks collected), 529 Haemaphysalis flava (34.73%), and one Haemaphysalis phasiana (0.07%). The highest number of free-living ticks in different habitats was collected in suburban parks (50), followed by those around rural natural villages (47) and barren grasslands (33), and then in forest areas (2). Parasitic ticks were collected from goats, dogs, and hedgehogs, which accounted for 55.79% (776 ticks), 9.35% (130), and 34.87% (485) of the 1 391 parasitic ticks collected, respectively. Homology analysis showed that individuals of the same tick species originated from the same evolutionary branch.

Conclusions Three tick species, H. longicornis, H. flava, and H. phasiana, were identified in Nanjing city. Haemaphysalis longicornis was the dominant species, and H. phasiana was identified as a newly monitored tick species in the region. Tick species composition varied among habitats and animal hosts. No significant genetic variation within tick species was observed.