Mesenchymal stem cells are considered potent sources that can be used for tissue regeneration and treatment of various diseases and syndromes. Among the mesenchymal stem cells, amniotic fluid-derived stem cells come forward as they possess some pluripotent properties and there are no serious ethical concerns upon their derivation. Therefore, gaining a better understanding of the nature of amniotic fluid stem cells is important. Although it is known that stem cells show slightly different characteristics between passages, scientists often consider only cell numbers and the proliferation pattern of the cells when deciding the passage to use in their studies. In this study, it was aimed to characterize rat amniotic fluid-derived stem cells for their mesenchymal and pluripotent features and make inter-passage comparisons by real-time qPCR to reveal their distinctions between different passages, and eventually help decide the appropriate passage numbers to be used in future research. The outcomes of the study showed that using rat amniotic fluid-derived stem cells at P5 could be beneficial for mesodermal differentiation studies and using them in earlier passages may be more favorable for the studies requiring better ectodermal differentiation properties.