SummaryIn this work a chromosomal study inEquus asinusis presented. Forty‐two specimens, belonging to five different Spanish breeds in risk of disappearing, have been analysed. Metaphases have been sequentially stained (uniform, G‐banded and C‐banded) and analysed. All specimens, except one, presented 2n = 62 chromosomes. Variation in chromosome number (2n = 63) is because of a fission in one chromosome of pair no. 3, resulting in two acrocentric chromosomes. Polymorphisms affecting the size of heterochromatic C+ bands and the presence of hetrochromatic 1p and 4p arms are also analysed. The results show no polymorphisms which can be attributed to a particular breed. From the chromosomal point of view, all Spanish asinine specimens correspond to a single population. Rearrangements detected suggest a fission tendency in the homologousE. asinusno. 3 chromosome in Equidae species.