<p>Most of the germplasm in the US Potato Genebank is stored as seedlots with varying levels of genetic diversity both within and among them. Therefore, we aim to determine the most efficient method for measuring this diversity. A seedlot from each of four species of tuber-bearing <i>Solanum</i> representing different ploidies and breeding systems (<i>S</i>. <i>fendleri</i>, <i>jamesii</i>, <i>microdontum</i>, and <i>sucrense</i>), was sown to produce 24 individuals. Additionally, three types of bulk samples were created. These samples were genotyped using Genotyping By Sequencing (GBS) with enhanced read depth (&gt; 100 × per sample). We introduced the shorthand term “Cap2” for loci where the two SNP alleles, known to be present among individuals, were detected in the bulk (<i>i.e.</i>, Captured). After excluding loci with missing calls or one fixed allele, 100% of the remaining loci were considered potentially Cap2. Cap2 was evaluated for loci categorized by different combinations of average minor allele frequencies (MAF) among individuals and read depth of the bulks. For loci with at least 5% MAF and a minimum of 100 reads, the average Cap2 for individuals was 81%, while all bulks, irrespective of species or bulking method, showed a Cap2 bulk average of 100% (average of 8,000 loci with an average of 144 reads). While genotyping a large number of individuals in a seedlot provides a precise measure of diversity, bulking DNA of many individuals followed by assessment of only the high-read loci with a MAF of at least 5% offers an efficient and optimized method for detecting the genetic landscape of a population with just a single sample.</p>