The objective of this research was to characterize the genetic diversity in 5 Spanish sheep breeds from high throughput data. We have used the OvineSNP50 Genotyping BeadChip to genotype 54,241 single nucleotide polymorphisms (SNPs) in 5 endangered Spanish ovine breeds (Xisqueta, Ripollesa, Roja Mallorquina, Canaria de Pelo o Pelibuey and Gallega) with the intention of investigating their genetic relationships and population structure. Multidimensional scaling plot and Admixture analyses revealed that the 2 insular breeds, i.e., Canaria de Pelo (Canary Islands) and Roja Mallorquina (Majorca Island), are the 2 most differentiated populations. African influences and prolonged geographic isolation may partly explain these observations. In contrast, the 3 peninsular breeds showed a weak level differentiation (FST = 0.01–0.03). We have also investigated the variability of the 5 Spanish breeds mentioned above through a RNA-seq approach. More specifically, we sequenced muscle mRNA from pools of 10 individuals by using a Hiseq 2000 platform (Illumina). Our main goal was to find out which percentage of the variation of these populations is breed-specific. Around 300 million reads were obtained per breed, and approximately 70% of them were sucessfully mapped to the latest version of the sheep reference genome. The total number of SNPs retrieved per breed ranged from 147,262 SNPs (Gallega) to 368,661 SNPs (Xisqueta). Obviously, genotyping experiments in a broader sample of individuals will be necessary to confirm the population-specificity of these SNPs. As a whole, our data suggest that ovine breeds share a substantial amount of variability, probably due to the combined effects of recent divergence and gene flow. However, we have also obtained evidence that thousands of variants appear to segregate specifically in particular breeds, a finding that reinforces the need of making every effort to conserve these unique genetic resources.