Domestic cats have several features that make them ideal vehicles for interspecies transmission of influenza viruses; however, they have been largely overlooked as potential reservoirs or bridging hosts. In this study we conducted serological surveillance to assess the prevalence of novel pandemic H1N1 as well as seasonal human influenza virus infections in domestic cats in Ohio, USA. Four-hundred serum samples collected from domestic cats (September 2009 - September 2010) were tested using a hemagglutination inhibition (HI) test. Seroprevalence of pandemic H1N1, seasonal H1N1 and H3N2 were 22.5%, 33%, and 43.5% respectively. In addition, a significant association between clinical feline respiratory disease and influenza virus infection was documented. In this sample of cats, prevalence of pandemic H1N1 did not follow the seasonality pattern of seasonal H1N1 or H3N2 influenza, similar to observations in humans. Pandemic H1N1 seroprevalence did not vary in relation to ambient temperature changes, while the seroprevalence of seasonal H3N2 and H1N1 influenza viruses increased with the decline of ambient temperature. Our results highlight the high prevalence of influenza infection in domestic cats, a seasonality pattern comparable to that of influenza in humans, and an association of infection with clinical respiratory disease.