To manage germplasm collections, molecular markers are becoming more prominent for modern genebanks. The understanding of the distribution of genetic diversity in populations and among species is crucial for utilization of the collections. Here, we describe the development of a DArT platform for a new oilseed crop, lesquerella (Physaria fendleri) of the Brassicaceae family as a model to characterize a collection in the National Plant Germplasm System (NPGS) with relatively little known in regards to the genetic diversity and traits. Using a subset of the Physaria germplasm conserved ex situ consisting of 86 accessions representing 12 species, a total of 2833 polymorphic markers were found with an average genotype call rate of 98.4% and a scoring reproducibility of 99.7%. Cluster analysis and principal coordinate analysis indicated that the different accessions were successfully classified by the marker system based on species, by geographical source, and breeding status. In the germplasm set analyzed, we observed that a substantial amount of variation exists with mean similarity among accessions at only 66%. Additional representative accessions will be genotyped to complete the representation from the entire P. fendleri collection. In addition to the microarray DArT platform, we also applied similar genome complexity reduction methods to develop SNP and DArT markers from short sequence reads from NGS platforms. The new platform resulted in over 15,000 high quality markers which revealed a similar picture of diversity as the microarray markers. These markers are expected to be also useful in crop breeding to supplement the available microsatellite markers in the taxa and hopefully encourage similar analysis of diversity in other minor and new oilseed crop species.