Walking catfish (Clarias macrocephalus) is important in both fisheries and aquaculture. Morphological characteristics of the species can change to adapt to living environments. The objective of this study was to quantify morphological variation of walking catfish in different environmental conditions. Morphological differences between 3 cultured and 3 wild walking catfish populations collected in Ca Mau, Can Tho, and Long An provinces were investigated by multivariate analyses of twenty-four morphometric and six meristic traits. Statistical analyses of conventional morphometric measures showed a divergence between the two groups of populations while meristic counts varied in similar ranges. Twenty morphometric characters (83.3%) were significantly different between cultured and wild populations. In which, eighteen morphometric charcters of wild population were significantly larger than cultured walking catfish. The morphometric differences between populations may be attributed by genetic differences or/and environmental factors. Results also showed that the relative gut length (RGL) of cultured populations (1.23±0.24) was significantly higher than that of wild walking catfish populations (1.04±0.25), indicating an adaptation to feed and feeding types of cultured fish in captive conditions.