SummaryThe novel mutation named ru2d/Hps5ru2‐d, characterized by light‐colored coats and ruby‐eyes, prohibits differentiation of melanocytes by inhibiting tyrosinase (Tyr) activity, expression of Tyr, Tyr‐related protein 1 (Tyrp1), Tyrp2, and Kit. However, it is not known whether the ru2d allele affects pheomelanin synthesis in recessive yellow (e/Mc1re) or in pheomelanic stage in agouti (A) mice. In this study, effects of the ru2d allele on pheomelanin synthesis were investigated by chemical analysis of melanin present in dorsal hairs of 5‐week‐old mice from F2 generation between C57BL/10JHir (B10)‐co‐isogenic ruby‐eye 2d and B10‐congenic recessive yellow or agouti. Eumelanin content was decreased in ruby‐eye 2d and ruby‐eye 2d agouti mice, whereas pheomelanin content in ruby‐eye 2d recessive yellow and ruby‐eye 2d agouti mice did not differ from the corresponding Ru2d/‐ mice, suggesting that the ru2d allele inhibits eumelanin but not pheomelanin synthesis.