Knowledge of karyotypical characteristics of a species is essential for understanding how sexually selected and sexually antagonistic traits evolve. The yellow dung fly Scathophaga stercoraria L. (Diptera: Scathophagidae) is an established model system for studies of sexual selection and sexual conflict, but karyotypical data are lacking to date. Here, the karyotype of S. stercoraria was characterized using conventional Giemsa-staining and C-banding techniques. The diploid chromosome set consists of 6 pairs of bi-armed meta- or submetacentric chromosomes. The sex chromosomes are the largest chromosomes and constitute 30% of the total length of the diploid set in females and about 25% in males. Males are the heterogametic sex, and the length of the Y chromosome is about three-quarters of that of the X chromosome. C-banding revealed that both sex chromosomes are largely heterochromatic. In contrast, in the five autosome pairs, heterochromatin is limited to narrow bands in the centromeric regions. This karyotypic information will help provide a more profound understanding of the inheritance of phenotypic variation in reproductive traits and the chromosomal locations of underlying genes.