Abstract The present research focuses to examine the meristic and landmark-based morphometric variations of barred spiny eel Macrognathus pancalus from four populations namely Dhakuria beel, Jessore (DBJ); Bohnni boar, Gopalgonj (BBG); the Arial kha river, Madaripur (AKRM) and the Nabaganga river, Jhenidah (NRJ) in Bangladeshi freshwaters. Six meristic counts were compared among four populations and significant differences were observed in number of caudal fin rays and number of pelvic fin rays in all meristic characters. The truss protocol was used in the present study based on six general morphometrics and fifteen truss measurements. Univariate statistics showed that five (standard length (SL), upper jaw length (UJL), lower jaw length (LJL), head depth (HD) and inter orbital (IO)) of six morphometric and eleven truss networks (viz. 2-3, 3-4, 5-6, 6-7, 7-8, 1-8, 2-8, 3-8, 3-7, 4-7 and 5-7) among fifteen truss measurements differed significantly. Cannonical discriminate function analyses were conducted among samples and the populations were fully intermingled. Pooled within-group correlation showed morphometric and landmark measurements; the first discriminant function (DF) accounted for 79%, the second DF accounted for 15.9% and third DF accounted for 5.1% of group variability. An euclidian dendrogram was prepared based on morphometric and truss measurements in four populations, where two clusters were mainly formed, in which first cluster formed by NRJ population was fully separated, and the second cluster was formed by two populations of AKRM and DBJ. Additionally, a subcluster BBG was formed with AKRM. On the basis of morphometric and truss measurements, 68.8%, 83.6%, 55.6% and 80% of original grouped cases were correctly classified in NRJ, AKRM, DBJ and BBG, respectively. These results specify the presence of different stocks of fish from four aquatic habitats. This study is highly significant for proper conservation and effective management of M. pancalus populations as well as for the scientific community for further research.