The present study aimed to compare different indirect and direct diagnostic techniques to diagnose Toxoplasma gondii in free-range chickens. Samples of 386 chickens obtained from 24 Paraná properties were used for serological analysis by indirect fluorescent antibody test (IFAT), modified agglutination test (MAT), and enzyme-linked immunosorbent assay (ELISA). Animals positive by IFAT and/or MAT had their tissues submitted to the mouse bioassay, and those who were positive in this technique had their blood, tissues, and acidic pepsin tissue digestion submitted to PCR (conventional, nested, and quantitative-PCR (qPCR)). One hundred and nineteen chickens (30.8 %) were positive in at least one of the serological tests, being 102 (26.4 %) in the IFAT, 64 (16.6 %) in the MAT, and 62 (16.0 %) in the ELISA. The IFAT was used as a gold standard, and the MAT showed higher sensitivity (46.0 %) and specificity (94.0) compared to ELISA (43.5 % and 93.6 %, respectively). Ninety samples of eighteen chickens positive in the mouse bioassay were subjected to PCR, and according to molecular tests, the conventional PCR detected the T. gondii DNA in 30 % (27/90) of the samples, in 38.8 % (35/90) with nested-PCR and 40.0 % (36/90) with real-time. According to molecular analyzes, the sensitivity was higher in ITS1 nested-PCR (69.4 %) and specificity in conventional PCR-529bp (90.7 %), using the qPCR as the gold standard. MAT and ELISA had similarities in concordance analyzes. The IFAT was the serological technique with the highest agreement with the mouse bioassay, and serological tests in parallel showed to be a good screening option for the isolation of T. gondii in chick tissues. The PCR markers effectively detected the parasite DNA, and the heart was the tissue with the highest number of positives samples. The conventional PCR had sensitivity similar to nested-PCR and qPCR and could be a cheaper alternative to diagnose T. gondii infection in chicken tissues.