In this work a chromosomal study in Equus asinus is 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 homologous E. asinus no. 3 chromosome in Equidae species.