Toxoplasma gondii is an obligate intracellular parasite recognized as the etiological agent of Toxoplasmosis, a zoonotic disease endemic in different countries worldwide. In Mexico, the disease is recognized as a human and animal health problem. T. gondii sporozoites have the capacity of been released from the gut lumen and infect several tissues, including kidneys. Rodents have been identified as intermediate hosts of T. gondii; nevertheless, no previous reports exist in Yucatan, Mexico, about their role in the infectious cycle. A total of 187 specimens (130 Mus musculus and 57 Rattus rattus) were studied. Total DNA was extracted from renal tissue and was utilized in the identification of T. gondii with the end-point Polymerase Chain Reaction (PCR) method. Six positive products were purified and sequenced in order to confirm and validate the results through the BLAST Sequence Analysis Tool. The PCR provided a global positivity of 5.34% (10/187). The BLAST analysis found a direct homology for T. gondii with identities and coverage of 96-98% and 98-100%, respectively. Our results confirm the role of M. musculus and R. rattus in the biological cycle of T. gondii and offer knowledge of the parasite transmission in intermediary hosts of the region.