Black flies (Diptera: Simuliidae) are distributed throughout the world, with more than 2200 formally described species. The family is renowned for its high frequency of cryptic species, offering an opportunity for integrative taxonomy, based on morphological, chromosomal, and molecular approaches. The biodiversity within Simulium (Psilopelmia) ignescens and S. (Psilopelmia) tunja in Colombia was estimated from the larval stage; 10 morphoforms were recognized based on 7 structural characters. This remarkable morphological variation was evaluated through 23 markers on the polytene chromosomes. We established 1 new cytoform in each nominal species. The congruence of the morphological and chromosomal assignments was evaluated using the mitochondrial marker Cytochrome Oxidase subunit I (COI) for each morphoform. The molecular data supported the chromosomal recognition of cytoforms (i.e., cryptic species). We also established the suitability of the COI marker for linking the pupal stage with each cytoform. Our results reveal the presence of hidden biodiversity in S. ignescens and S. tunja and demonstrate the power of polytene chromosomes as a tool for evaluating simuliid diversity, while illustrating the importance of integrated analyses in modern taxonomy.