In this study, the mitochondrial DNA diversity of six Spanish donkey breeds and two African donkey populations (one from Morocco and the other from Zimbabwe) was analysed. A total of 79 animals were sequenced for 313 bp of the cytochrome b gene, and 91 individuals for 383 bp of the D-loop region or control-region. Sequence comparisons and phylogenetic analyses of both Spanish and African populations revealed low diversity. Only six and seven haplotypes respectively were found in cytochrome b and the D-loop region. Relatively low nucleotide diversity (π) values were detected in these populations. The π values, from the D-loop region, ranged from 0.0006 to 0.0169 for the Catalana and Andaluza breeds, respectively. The obtained results seem to confirm the existence of two divergent maternal lineages of African origin (Equus asinus africanus and E. a. somaliensis). In this paper the genetic relationships between these breeds are analysed and compared with those obtained in other European populations. Also, the data on the genetic relationships between the populations, obtained from nuclear DNA (microsatellites) and mitochondrial DNA (cytochrome b and D-loop region) is argued and interpreted.