Purpose: <p>This study investigated the morphology of isthmus in mandibular first molar of a Malaysian subpopulation.</p> Method: <p>A total of 140 double-rooted mandibular first molars were scanned using micro-computed tomography. Fan et al. classification was used to classify the isthmus morphology. The chi-square test was used to compare the qualitative variables. The independent samples t-test and Mann Whitney U test were used to compare the quantitative variables.</p> Results: <p>The prevalence of isthmus was 77.1%. The mesial root had the majority of isthmuses [135 (89.4%)]. Of these, 65.2% were “Mixed” type. The distal roots had only 16 (10.6%) of all isthmuses. Of these, 12 (75%) were “Mixed” type. No significant difference in the type of isthmus between the mesial and distal roots (<i>p</i> = 0.335). However, a significant association was found between root type and the presence of isthmus (<i>p</i> &lt; 0.001). There was no significant difference neither in the volume of canal part with isthmus (<i>p</i> = 0.703 for sheet and 0.149 for mixed types) nor in the percentage volume (<i>p</i> = 0.601 for sheet and 0.299 for mixed types) between the mesial and distal roots in sheet and mixed isthmus types.</p> Conclusion: <p>In this population, the mandibular first molar showed a high complexity in the isthmus anatomy, especially in the mesial root.</p>