Indigenous cattle play a vital role in subsistence and livelihood of pastoral producers in Eritrea. In order to optimally utilize and conserve these valuable indigenous cattle genetic resources, the need to carry out an inventory of their genetic diversity was recognized. This study assessed the genetic variability, population structure and admixture of the indigenous cattle populations (ICPs) of Eritrea using a genotype by sequencing (GBS) approach. The authors genotyped 188 animals, which were sampled from 27 cattle populations in three diverse agro-ecological zones (western lowlands, highlands and eastern lowlands). The genome-wide analysis results from this study revealed genetic diversity, population structure and admixture among the ICPs. Averages of the minor allele frequency (AF), observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (FIS) were 0.157, 0.255, 0.218, and -0.089, respectively. Nei’s genetic distance (Ds) between populations ranged from 0.24 to 0.27. Mean population differentiation (FST) ranged from 0.01 to 0.30. Analysis of molecular variance revealed high genetic variation between the populations. Principal component analysis and the distance-based unweighted pair group method and arithmetic mean analyses revealed weak substructure among the populations, separating them into three genetic clusters. However, multi-locus clustering had the lowest cross-validation error when two genetically distinct groups were modelled. This information about genetic diversity and population structure of Eritrean ICPs provided a basis for establishing their conservation and genetic improvement programmes.