A Holstein-Fresian calf with multiple congenital malformations was subjected postmortem to anatomical and genetic investigation. The calf was small (20 kg), had shortened limbs and was unable to stand up. It lived only 44 days. Detailed anatomical investigation revealed the following features: head asymmetry, the relocation of the frontal sinus and eye orbits, hypoplastic thymus without neck part, ductus Botalli, unfinished obliteration in umbilical arteries, and a bilateral series of tooth germs in the temporal region. Cytogenetic examination, performed on in vitro cultured fibroblasts, showed a unique mosaic karyotype with a marker chromosome-60,XX[9 2%]/60,XX,+mar[8%], which was for the first time described in cattle. No other chromosome abnormalities indicating chromosome instabilities, like chromatid breaks or gaps were identified, thus teratogenic agent exposure during pregnancy was excluded. The marker chromosome (mar) was small and it was not possible to identify its origin, however, sequential DAPI/C (4',6-diamidino-2-phenylindole) band staining revealed a large block of constitutive heterochromatin, which is characteristic for centromeric regions of bovine autosomes. We suppose that the identified marker chromosome was a result of somatic deletion in an autosome and its presence could be responsible for the observed developmental malformations. In spite of the topographic distance among the affected organs, we expected a relationship between anatomical abnormalities. To the of our best knowledge, this is the first case of a mosaic karyotype with a cell line carrying a small marker chromosome described in a malformed calf.