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Toxoplasma gondii is an intracellular parasite that can infect all warm-blooded animals including humans. Recent studies showed that T. gondii strains from South America are genetically diverse. The present work aimed to determine T. gondii prevalence in free-ranging chicken in northwest Parana state in Brazil by two serological tests, to isolate the parasites from seropositive chickens and to genotype the isolates. Antibodies to T. gondii in 386 serum samples from 24 farms were investigated by immunofluorescence antibody assay (IFA) and modified agglutination test (MAT). Samples having titers ≥ 16 were considered positive for both tests. Among the 386 serum samples, 102 (26.4%) were positive for IFA, 64 (16.6%) were positive for MAT, 47 (12.2%) were positive in both tests, and 119 (30.8%) were positive in at least one of the two tests. Brain and pool of heart, lung, and liver from the 119 seropositive chickens were used for mouse bioassay to isolate the parasites. Thirty eight (31.9%) of these seropositive chickens were considered positives in mouse bioassay and 18 isolates were obtained. The isolates were characterized by 10 PCR-RFLP genetic markers including SAG1, SAG2 (5'-3'SAG2, alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Results of genotyping were compared with the genotypes in ToxoDB database. It revealed ten genotypes, including ToxoDB PCR-RFLP genotypes #6 (n = 2), #19 (n = 1), #21 (n = 2), #111 (n = 2), #152 (n = 1), and #175 (n = 1) and four new types not described before. Our results confirmed a high genetic diversity of this parasite in southern Brazil and also showed that the use of two serological tests in combination can improve the chance of T. gondii isolation. More studies should be taken to determine the zoonotic potential of chickens in the transmission of T. gondii.