The karyotype and cytogenetic features of darkling beetle Accanthopus velikensis were analysed using conventional and differential staining. The diploid number was determined as 2n = 20 and the presence of Xy(p) sex determination system was observed with DAPI and silver staining as well as conventional staining. Although a single nucleolar material was observed in prophase I nuclei, multiple argyrophilic signals in diakinesis-metaphase I plates makes it difficult to determine the exact NOR location. Both conventionally and differentially stained plates showed that heterochromatin is mostly concentrated on centromeric regions of A. velikensis chromosomes. Obvious telomeric signals on some rod shaped bivalents as well as the X chromosome were also detected with AgNO3 and DAPI staining. Although presented karyotype of A. velikensis resemble to those of other Helopini members and follows the common patterns of Tenebrionid karyotypes, slight differences in chromosome morphologies, NORs and the heterochromatin distribution were detected. Our specimens also showed a unique haplotype for COI sequences with an 84-83% sequence similarity to database sequences for Tenebrionidae.