The phenotype of an organism not only reflects its genome, but also it can display its habitat and life style features. Hence, this study was aimed to apply the landmark-based geometric morphometric method in the study of the phenotypic plasticity of killifish (A. sophiae) from Cheshme-Ali Damghan and Shour river of Eshtehard. A total of 86 specimens were sampled from Cheshme-Ali (52 specimens) and Shour river (34 specimens). The left side of each specimen was photographed using a digital camera and fifteen landmark points were digitized on 2D images using TpsDig2. The landmark data of the two sexes of both populations were analyzed separately using DFA and T-test Hotelling after GPA superimposition. The patterns of body shape differences between the two sexes of both populations were illustrated. The results revealed significant differences between the body shape of two sexes in both populations (P<0.001). The morphological difference patterns between the two sexes were found in the depth of the body and caudal peduncle and the position of the pectoral fin. The findings of this study suggest that different characteristics of the habitats have driven changes in the morphological attributes of the inhabitant killifish populations along with geographical separation as evolutionary factors.