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Abstract This study was undertaken to determine the genetic structure, evolutionary relationships, and the genetic diversity among 18 local cattle breeds from Spain, Portugal, and France using 16 microsatellites. Heterozygosities, estimates of Fst, genetic distances, multivariate and diversity analyses, and assignment tests were performed. Heterozygosities ranged from 0.54 in the Pirenaica breed to 0.72 in the Barrosã breed. Seven percent of the total genetic variability can be attributed to differences among breeds (mean Fst = 0.07; P < 0.01). Five different genetic distances were computed and compared with no correlation found to be significantly different from 0 between distances based on the effective size of the population and those which use the size of the alleles. The Weitzman recursive approach and a multivariate analysis were used to measure the contribution of the breeds diversity. The Weitzman approach suggests that the most important breeds to be preserved are those grouped into two clusters: the cluster formed by the Mirandesa and Alistana breeds and that of the Sayaguesa and Tudanca breeds. The hypothetical extinction of one of those clusters represents a 17% loss of diversity. A correspondence analysis not only distinguished four breed groups but also confirmed results of previous studies classifying the important breeds contributing to diversity. In addition, the variation between breeds was sufficiently high so as to allow individuals to be assigned to their breed of origin with a probability of 99% for simulated samples.