Cats are susceptible to infection with severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Whilst a number of studies have been performed worldwide on owned cats, limited data are available on stray, colony or shelter cats. We investigated SARS-CoV-2 infection in a stray cat population before and during human outbreaks of SARS-CoV-2 in cities in the Lombardy region in northern Italy, a high endemic region for SARS-CoV-2, using serological and molecular methods. A cohort of different samples were collected from 241 cats, including frozen archived serum samples from 136 cats collected before the 2019 coronavirus disease (COVID-19) pandemic and serum, pharyngeal and rectal swab samples from 105 cats collected during the SARS-CoV-2 outbreak. All pre-pandemic samples tested seronegative for antibodies against the nucleocapsid of SARS-CoV-2 using indirect enzyme linked immunosorbent assay (ELISA) test, while one serum sample collected during the pandemic was seropositive. No serological cross-reactivity was detected between SARS-CoV-2 antibodies and antibodies against feline enteric (FECV) and infectious peritonitis coronavirus (FIPC), Feline Immunodeficiency Virus (FIV), Feline Calicivirus (FCV), Feline Herpesvirus-1 (FHV-1), Feline Parvovirus (FPV), Leishmania infantum, Anaplasma phagocytophilum, Rickettsia spp., Toxoplasma gondii or Chlamydophila felis. No pharyngeal or rectal swab tested positive for SARS-CoV-2 RNA on real time reverse transcription-polymerase chain reaction (rRT-PCR). Our data show that SARS-CoV-2 did infect stray cats in Lombardy during the COVID-19 pandemic, but with lower prevalence than found in owned cats. This should alleviate public concerns about stray cats acting as SARS-CoV-2 carriers.