Snakes represent a taxonomically diverse group that plays a critical role in ecosystems and is associated with public health issues; however, accurate identification remains challenging in many resource-limited settings. We evaluated microscopic ultrastructures in the microdermatoglyphics of scales collected from shed-off skin as a non-invasive and cost-effective diagnostic method. Shed-off skins from twelve adult specimens—three each from two venomous cobras (Naja naja, Naja kaouthia) and two non-venomous colubrids (Ptyas mucosa, Fowlea piscator)—were collected from different geolocations in Bangladesh. Then, 15 types of scales representing head, body, and tail regions of snakes were collected and prepared for microscopic observation, and the micro-ornamentation of microdermatoglyphics was examined at 40X, 100X, and 400X magnifications. Six ultrastructural characters (edge perimeter, edge area, edge WPD, follicle perimeter, follicle area, follicle 20,000 µm²) in microdermatoglyphics were quantified. Paired t-tests and Wilcoxon signed-rank tests (p = 0.001) assessed stability within species and variation between species, as well as group. The results show that 75.56%, 58.89%, 76.67%, and 77.78% of characters are stable across the 15 scales in N. naja, N. kouthia, P. mucosa, and F. piscator, respectively. Four characters (out of six) significantly (p = 0.001) differentiate N. naja from N. kaouthia, whereas all six characters separate P. mucosa from F. piscator. Furthermore, five characters significantly differentiate more than 50% of the analyzed scales of the venomous from the non-venomous groups. PCA accounted for 77.6% (PCA1 and PCA2) of the variance, with clear species-wise and group-wise clustering. This approach enables reliable differentiation between closely related congeneric species and far distant species, providing a practical alternative to conventional taxonomic techniques. It holds potential for application in field-based diagnostics, snakebite management, forensic herpetology, and biodiversity monitoring.