Lentil (Lens culinaris ssp. culinaris) is a nutritious and affordable pulse with an ancient crop domestication history. The genus Lens consists of seven taxa, however, there are many discrepancies in the taxon and gene pool classification of lentil and its wild relatives. Due to the narrow genetic basis of cultivated lentil, there is a need towards better understanding of the relationships amongst wild germplasm to assist introgression of favourable genes into lentil breeding programs. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows multiplexing of up to 384 samples or more per library to generate genome-wide single nucleotide Polymorphism (SNP) markers. In this study, we aimed to characterize our lentil germplasm collection using a two-enzyme GBS approach. We constructed two 96-plex GBS libraries with a total of 60 accessions where some accessions had several samples and each sample was sequenced in two technical replicates. We developed an automated GBS pipeline and detected a total of 266,356 genome-wide SNPs. After filtering low quality and redundant SNPs based on haplotype information, we constructed a maximum-likelihood tree using 5,389 SNPs. The phylogenetic tree grouped the germplasm collection into their respective taxa with strong support. Based on phylogenetic tree and STRUCTURE analysis, we identified four gene pools, namely L. culinaris/L. orientalis/L. tomentosus, L. lamottei/L. odemensis, L. ervoides and L. nigricans which form primary, secondary, tertiary and quaternary gene pools, respectively. We discovered sequencing bias problems likely due to DNA quality and observed severe run-to-run variation in the wild lentils. We examined the authenticity of the germplasm collection and identified 17% misclassified samples. Our study demonstrated that GBS is a promising and affordable tool for screening by plant breeders interested in crop wild relatives.