The Y-chromosome of mice has a crucial role in sex determination, gender ratio equilibrium as well as male fertility, and is moreover involved in behavioral, immunological, and cardiovascular traits. During routine short tandem repeat genotyping of C57BL/6 substrains, a unique deletion on the Y-chromosome long arm of males from the commercially available inbred substrain C57BL/6JBomTac was identified. In this study, the deletion was confirmed by fluorescence in situ hybridization on metaphase spreads and the extent of the deletion was assessed using position-specific genetic markers. It covers 40 Mbp of the Y-chromosome long arm, ranging from at least 6.57 to 46.73 Mbp. Therefore, C57BL/6JBomTac might be a valuable model system for Y-chromosome research. A deletion spanning almost half of the Y-chromosome long arm should not be neglected regarding the evaluation of scientific experiments. Our data are in line with others that it is of major importance that the usage of mice strains requires the exact nomenclature including the name of the substrain.