The validation of an association between a genotype and a seed metabolic trait can be a time-consuming process. Strategies to speed this process are needed in this age of rapid and high throughput genomics. In this study, we have developed a protocol to associate the fatty-acid composition of a single pennycress seed as a phenotypic trait to a genotype. We use an approach that allows for parallel assessment of genotypes and fatty acid composition on a small single seed of field pennycress. We were able to show that fatty acid values predicted from a single seed are similar to the values of pooled seeds for linoleic acid (17.32% vs 18.10%), linolenic acid (11.10% vs 11.86%) and erucic acid (38.43% vs 38.74%) with the exception of oleic acid (10.79% vs 12.44%) and eicosenoic acid (8.56% vs 10.64%) based on the pair-wise Tukey's test at a p-value of 0.05. We were also able to show that DNA can be extracted from the single defatted seeds and the DNA can be used for SNP genotyping. This approach was tested on two independent F2 segregating populations to demonstrate linkage between fatty acid composition and the respective genotypes. The F2 populations for this study were derived from crosses among the mutants of Fatty Acid Elongation 1 (Ta FAE1), Reduced Oleate Desaturase 1 (Ta ROD1) and Fatty Acid Desaturase 2 (Ta FAD2). We assessed fatty acid composition of single F2 seeds from each of the genotype classes in the segregating populations identified using allele-specific markers. We found that mutations in Ta FAE1 and Ta FAD2 had semi-dominant effect, and that the mutation in Ta ROD1 was recessive. Within each population, doubly homozygous single seeds for Ta fae1-1 and Ta rod1-1 or Ta fae1-1 and Ta fad2-2 created the most extreme fatty acid alteration compared to wild type. F2 single seeds genotyped as double mutants of Ta fae1-1/ Ta rod1-1 from the first population averaged 56.42% oleic acid compared to under 15% for wild type along with little to no detectable erucic acid compared to over 35% in wild type. F2 single seeds genotyped as double mutants of Ta fae1-1/Ta fad2-2 from the second population averaged 85.87% oleic acid with significant reduction in linoleic, linolenic, eicosenoic and erucic acid. Fatty acid compositions of these double mutants were further confirmed using a pool of seeds. This study demonstrates the rapid assessment of metabolites and genotypes, thus helping us characterize fatty acid genes in pennycress. Application of such methods to a species with small seeds like pennycress will increase the efficacy of functional genomics and can be applied towards other metabolite traits.