Objective: An assessment of the prevalence of Toxoplasma gondii infection in cats from southwestern Poland using serology, coproscopy and PCR methods. Material and methods: In total, 208 cats (139 females and 68 males), aged 0.5–12 years (mean=2.6) from 25 localities in southwestern Poland were examined by indirect immunofluorescence assay (IFAT) to estimate the T. gondii serological status. Faecal samples of 41 cats were examined for the presence of oocysts/DNA T. gondii by microscopy and Real-time/nested PCR. After flotation (with NaNO3), pellets from faecal samples were disrupted by 10 cycles of freezing (liquid nitrogen) and warming. DNA was extracted using QIamp DNA Stool Mini Kit (Qiagen), according to the manufacturer’s instruction. Results: The positive results in IFAT for anti-T. gondii IgG and IgM antibodies were found in 143 of 208 tested cats (68.8%). Among positive results, 14.5%, 34.1% and 51.4% were detected in titre ranges 128–512, 1,000–2,000 and ≥ 4,000, respectively. In 23.1% of cat sera anti-T. gondii IgM antibodies were found. The prevalence of anti-Toxoplasma antibodies was significantly greater in older cats (>1 year) (83.5%) than in younger cats (48.3%) (P<0.05), in females (74.1%) than in males (58.8%) (P<0.05), and in cats kept outdoors than indoors (69.7% vs. 16.7%) (P<0.01). Among the 41 faecal samples examined, the presence of structures resembling T. gondii oocysts was found in 2 samples (4.9%), and for one of these samples (2.4% of the total) the result was also confirmed by PCR. Conclusions: T. gondii infection in domestic cats is highly prevalent in southwestern Poland. Information on the prevalence of infection in cats can be useful for assessing T. gondii environmental contamination and the risk for public health.