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Abstract This study aimed to develop direct PCR methods, which enable the diagnosis of brucellosis agents from ruminant aborted fetus samples at species and genus levels, and determine the applicability of the newly developed methods. For this purpose, 137 lung, 137 liver, and 52 fetal stomach fluid samples belonging to 166 ruminant aborted fetuses (326 samples in total) were examined. Firstly, agent isolation and identification were performed and species-specific multiplex PCR (m-PCR) from the culture was applied to the samples. In addition, the Mayer-Scholl m-PCR method was modified and termed 'modified Mayer-Scholl', and genus specific Bcsp31 PCR was also modified with minor changes. Four different methods were applied to direct examination samples and the obtained results were compared. The conventional culture method was set as the standard method to which sensitivities and specificities of the molecular methods were calculated. According to the assessments on the basis of fetus (n = 166), sensitivity and specificity values for modified Mayer-Scholl m-PCR method were 94.11% and 98.76%, and the same indicators for the modified Bcsp31 PCR were 95.29% and 98.76%, respectively. When all organ samples were taken into account (n = 326), sensitivity and specificity values for the modified Mayer-Scholl m-PCR method were 85.38% and 98.06%, and for the modified Bcsp31 PCR, they were 83.62% and 98.06%, respectively. As a result, it was found that the diagnostic power of the tests were 'high' when results were evaluated at fetus level. On the other hand, it was found to be 'clinically useful' when evaluated at organ level. We concluded that species level identifications can be made through the modified Mayer-Scholl method, which is a direct m-PCR method, with a high diagnostic power by specifying DNAs belonging to Brucella species directly from clinical samples. Highlights • Multiplex PCR is applicable to the direct diagnosis of brucellosis from clinical samples • The sensitivity and specificity values of the PCR are considered as satisfactory • For the diagnosis of brucellosis, first, the fetal stomach fluid should be preferred • Then, lung and liver tissues should be opted for diagnosis, respectively [ABSTRACT FROM AUTHOR]