Brucellosis is recognized as a major public health concern leading to critical economic losses in livestock animals. The present study assessed Brucella spp. isolated from aborted ovine and caprine fetuses in different parts of Iran between 2016 and 2019. It used classic and molecular methods in order to determine the Brucella species carrying higher risks of abortion complications in these animals. A total of 189 samples from 35 cases/case series from milk (16 sheep, and 8 goats), 19 abomasum content (sheep), and 146 aborted fetuses (116 sheep, and 30 goats) were bacteriologically examined. Subsequently, the resultant Brucella isolates were further characterized by phenotypic and molecular approaches. The multiplex Polymerase chain reaction (PCR) (Bruce-ladder) and IS711-based PCR were performed on all the extracted DNA to evaluate the presence of Brucella spp. As suggested by the obtained results, all recovered isolates from ovine and caprine abortion samples were either B. melitensis or B. abortus. An issue of concern was the implication of B. melitensis vaccine strain Rev1 in a small portion of sheep and goat abortion cases. Despite the recent B. abortus burden in ovine, aborted cases were predominantly associated with B. melitensis infections in both ovine and caprine, and B. melitensis biovar 1 was responsible for the majority of studied cases. These data and the techniques implemented in the present study can shed light on the level of implication of different Brucella species in ovine and caprine abortion in Iran. The present study identified Brucella agents responsible for abortion in small ruminants at the biovar level. Therefore, it provides precious information for future control programs and vaccination strategies in Middle Eastern regions.