Globally, toxoplasmosis is an important zoonotic parasite infection of many warm-blooded animals (including humans). Toxoplasma gondii oocysts are widespread, and their contamination can be primarily attributed to the members of the Felidae family. This study aimed to estimate the prevalence and determine the dense granule antigen 6 (GRA6) genotype of T. gondii among domestic cats in the Phayao Province, Thailand. A total of 124 fecal samples were collected from owned cats in the Muang district, Phayao, Thailand, from January to December 2020. Fecal samples were tested for the presence of T. gondii DNA using targeted B1 gene polymerase chain reaction (PCR) amplification, and positive samples were subsequently analyzed for their T. gondii genotype through PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing of the GRA6 gene. Among the 124 samples, 46 (37.1%) were tested positive for T. gondii. Only 10 positive DNA samples were successfully amplified for the GRA6 marker. Subsequent PCR-RFLP and sequence analyses indicated that all T. gondii isolates from cats in Phayao belonged to GRA6 genotype I. Data revealed that toxoplasmosis is remarkably distributed among (studied) domestic cats in Phayao, Thailand. Moreover, the virulent GRA6 allele was found to be circulated among domestic cats in this area. However, no significant correlation was observed between infection rates and different risk factors, which indicated that pet cats of any age, gender, or breed have similar risks of being infected with T. gondii. Our results further suggested that infective oocysts of T. gondii are widely distributed and that environmental contamination with these oocysts will introduce more risks of disease transmission to humans and other animals.