Mules (Equus asinus x Equus caballus) represent first-generation hybrids between a female horse (mare) and a male donkey (jack). They are generally considered to have first appeared north of the Alps with Roman influence, a time period in which written and iconographic sources support their key role for transport and traction, both in farming and the military. The archaeozoological evidence for mules is, however, contentious as faunal assem-blages are difficult to assign to either parental species or hybrids based on morphometric data alone. Here we leverage low-coverage DNA sequence data and Zonkey computational analyses to assess the occurrence of mules within Roman equid faunal assemblages in the alpine foreland. While morphological data previously assigned 17 remains to mules, successful DNA analysis of 12 remains revealed that 11 were in fact horses, one female and ten males. Eight mtDNA d-loop haplogroups were identified and genetic diversity within Roman equids corresponds to non-threatened modern local breeds. Two remains genetically identified as mules belonged to haplogroups F and I. Our results suggest that the importance of mules in the Roman archaeological record of the alpine foreland, and probably elsewhere, may have been previously over-estimated. Whether this is true for other regions of the Roman Empire needs to be evaluated. Further genomic testing for equid species and their hybrids and molecular sexing will improve our knowledge on this important issue.