Rodents and stray cats are the sources of many parasitic infections including for other animals and human. Toxoplasmosis has a wide range of laboratory factors in its intermediate and definite hosts. Regarding the importance of rodents and stray cats as the hosts that spread the , it is necessary to obtain comprehensive information about these animals in the life cycle of . The objective was to investigate the new prevalence of toxoplasmosis among target animals in Iran, using GRA6 gene in combinacion with ELISA avidity. In this study, 286 rodents and 210 stray cats were collected and their heart tissues extracted to obtain DNA, blood samples and IgG Ab of parasite. We detected the positive tissue samples in our study by the nested-PCR method. Then, we examined IgG ELISA avidity for assessment of toxoplasmosis among rodents and stray cats. This study, was conducted in January to March 2017, based on the prevalence study. The findings revealed that 246/286 (86.01%) of rodents and 180/210 (85.71%) of stray cats were positive by IgG ELISA avidity methods. moreover, 68 rodents samples and 38 stray cats samples were positive concerning the GRA6 gene; and these positive samples were at intermediate levels for IgG avidity. We concluded that the new prevalence of toxoplasmosis among rodents and stray cats was at high levels, using the serologic method in Northeast of Iran and the results of quantitative ELISA avidity were as the same as those of the nested-PCR for detecting recent toxoplasmosis in these hosts.