Simulium quinquestriatum Shiraki (Diptera: Simuliidae), a human-biting fly that is distributed widely across Asia, is a vector for multiple pathogens. However, the larval development of this species is poorly understood. In this study, we determined the number of instars in this pest using three batches of field-collected larvae from Guiyang, Guizhou, China. The postgenal length, head capsule width, mandibular phragma length, and body length of 773 individuals were measured, and k-means clustering was used for instar grouping. Four distance measures—Manhattan, Euclidean, Chebyshev, and Canberra—were determined. The reported instar numbers, ranging from 4 to 11, were set as initial cluster centers for k-means clustering. The Canberra distance yielded reliable instar grouping, which was consistent with the first instar, as characterized by egg bursters and prepupae with dark histoblasts. Females and males of the last cluster of larvae were identified using Feulgen-stained gonads. Morphometric differences between the two sexes were not significant. Validation was performed using the Brooks–Dyar and Crosby rules, revealing that the larval stage of S. quinquestriatum is composed of eight instars.