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• In the most of stray cats in Milan the seroprevalence for FCV is high, whereas less than 50% of cats were seropositives against FPV and FHV-1. • The feline calicivirus represents the most common circulating pathogen in Milan, as observed in other studies worldwide. • An increase of antibody protective titres from kitten to senior is generally observed for the three pathogens. The aim of the study was to determine the seroprevalence of feline panleukopenia virus (FPV), feline herpesvirus type 1 (FHV-1) and feline calicivirus (FCV) in stray colony cats from Milan, Italy. Cats were divided in groups based on age, gender, reproductive status, health status and colony of origin. Blood samples were tested with an in-clinic ELISA test. The possible presence of a link between the antibody titre or the presence of seropositive results and the independent variables (age, gender, reproductive status, health status and colony location) was assessed by means of multinomial and univariate logistic regression models, respectively. Seroprevalence of 85.4% was reported for FCV. The diffusion of the other two pathogens in the cat population was much lower compared to FCV, with 45.7% and 37.1% seroprevalence observed for FPV and FHV-1, respectively. An increase of antibody titres from kitten to senior was generally observed for the three pathogens. Age was a statistically significant variable for FHV-1, with senior cats significantly associated with higher antibody titres and higher percentages of seropositive animals compared to younger age groups. Neutered cats had significantly higher antibody titres and showed significantly higher FHV-1 seroprevalences compared to sexually intact cats. Colonies from two of the nine administrative districts of Milan showed significantly higher FPV seroprevalences compared to the others. No other significant differences were observed. Our results, based on cats belonging to 70 different colonies located in urban areas far from each other, suggest that the three viruses circulate in the feline population of stray cats in Milan. The feline calicivirus represents the most common circulating pathogen, as observed also in other studies worldwide. Finally, our results suggest that stray cats may be not adequately protected against FPV, FHV-1 and FCV and vaccination could be a possible strategic solution, especially for FPV. [ABSTRACT FROM AUTHOR]