Single nucleotide polymorphisms (SNPs) are becoming the most utilized markers in population genetics studies. Reduced representation genome techniques, as genotyping-by-sequencing (GBS), allow identifying great numbers of polymorphisms that are useful to analyze genetic diversity in non-model organisms. In this investigation, we implemented a GBS protocol and different bioinformatics tools to identify a novel SNP panel and investigate the genetic structure and variability in populations of little owl (Athene noctua) sampled from seven geographical regions in Europe (Balkans, north-west Europe, north, central and south Italy, Spain and Portugal). We obtained a total of 22,185 putative SNPs and 1306 indels. After the filtering procedure, 7175 SNP loci and 53 individuals met the stringent quality control measures. Neutrality test identified 281 candidate loci under positive selection, which were used for population genetic analyses. The selected SNP panel allowed an accurate description of the genetic structure of the little owl.