Locomotion is a complex trait directly linked to various components of fitness. Environmental factors influence type of locomotion and morphological traits associated with locomotion such as body size and appendage length. In subterranean environment the selection factors which influence the evolution of locomotion are occasionally opposing. In this study, we examined the interactions between locomotion, morphology and environment of selected amphipod species of the genus Niphargus living in different cave microhabitats (i.e., lakes, streams). We collected individuals of eight species and acclimated them in the cave laboratory of the Department of Biology. We measured their morphological traits supposedly associated with locomotion and made ethograms based on video recordings. We used idTracker 2.1 and R 3.5.1. to calculate the total path length, movement time, maximum and average speed. With principal component analysis we reduced the number of morphological and behavioral variables. To test how lake and stream species differ in morphology and locomotion we used MCMCglmm package in R. The correlation between morphology and locomotion was analyzed with regression analysis. We sought to determine whether morphology predicts locomotion. Lake species were larger, stouter and had longer appendages, while stream species were smaller, slimmer with shorter appendages. We have shown that stream species move lying on their side with slow crawling and tail-flipping movements, whereas lake species move faster and display different locomotion modes. One of the lake species almost exclusively moved in an upright or semi-upright position reminiscent of walking.