Toxoplasma gondii, a widespread parasite, poses significant public health concerns. It infects humans and animals, with rodents serving as important intermediate hosts. The present study investigated the prevalence and genetic ITS1 diversity of T. gondii in wild rodents from Lithuania. A total of 469 rodents from eight species were captured across various regions, and DNA from neural and cardiac tissues was analyzed using nested PCR. Overall prevalence of T. gondii was 26.2% (95% CI = 22.3–30.5). The prevalence of infection varied among rodent species (0–50.0%) and across geographic locations. A mere few rodents exhibited concurrent infections in both tissues examined. Toxoplasma gondii was detected more frequently in the brains of Apodemus flavicollis and hearts of Clethrionomys glareolus, and in the males of Microtus arvalis. A total of 19 distinct ITS1 genotypes were identified, including 17 novel ones; Genotype 1 was the most prevalent and widely distributed. Phylogenetic and network analyses revealed a star-like topology centered on Genotype 1 and confirmed the accurate identification of T. gondii in Lithuanian rodents. This study provides the first evidence of T. gondii in wild rodents in Lithuania, highlighting the need for further research on its prevalence and potential impact on public health and wildlife.