Objectives In this study, we evaluated the potential association between the habitat types of feral cats and theprevalence of selected infectious pathogens and health status based on a set of blood parameters.Methods We live-trapped 72 feral cats from two different habitat types: an urban area (n = 48) and a ruralagricultural area (n = 24). We compared blood values and the prevalence of feline immunodeficiency virus (FIV),feline leukaemia virus (FeLV) and haemotropic Mycoplasma infection in feral cats from the two contrasting habitats.Results Significant differences were observed in several blood values (haematocrit, red blood cells, bloodurea nitrogen, creatinine) depending on the habitat types and/or sex. Two individuals from the urban area wereseropositive for FIV (3.0%), and eight (12.1%) were positive for FeLV infection (five from an urban habitat andthree from a rural habitat). Haemoplasma infection was more common. Based on molecular analysis, 38 cats(54.3%) were positive for haemoplasma, with a significantly higher infection rate in cats from rural habitats (70.8%)compared with urban cats (47.8%).Conclusions and relevance Our study observed haematological and serum biochemical values, and prevalenceof selected pathogens in feral cat populations from two different habitat types. A subset of important laboratoryparameters from rural cats showed values under or above the corresponding reference intervals for healthydomestic cats, suggesting potential differences in the health status of feral cats depending on the habitat type. Ourfindings provide information about the association between 1) blood values (hematological and serum biochemistryparameters) and 2) prevalence of selected pathogen infections with different habitat types; this may be importantfor veterinarians who work with feral and/or stray cats and for overall cat welfare management.