Bacteria play a critical role in biogeochemical cycling and ecosystem function. Vibrio spp. are ubiquitous in near shore and pelagic environments. These bacteria are genetically plastic with increased ability to adapt to fluctuating environments. As DNA sequencing and bioinformatics approaches develop at an exponential rate, the function of many genes remains unknown. Therefore, high-throughput experimental testing of gene function is necessary. In this study, two ecotypes of fifteen Vibrio spp. were evaluated by genome analysis and by carbon utilization via Phenotypic Microarray Plates (MAPs). A clustering based analysis identified that the Vibrio species were not grouped by ecotype and there were discrepancies in the relationship between the phenotype and genotypes. I focused on the variation between two strains ED008 and ED582, which varied in annotated gene content by only 1.71%, but varied in unique carbon use by 40.85%. The analysis of the glucose pentaphosphate pathway identified pseudogenes in three strains which caused their negative phenotype. ED008 was the only strain that was able to grow on L–sorbitol. However, the genotype analysis showed that all strains lacked essential enzymes in the sorbitol pathway. Upon further analysis several substitutes were identified as potential transporters, but these were also in seven strains that showed no growth on the substrate. An extensive blast search identified two hypothetical proteins only present in ED008. Interproscan showed that one of the hypothetical proteins had membrane components and could be involved in the uptake of L-sorbose. My research offers a framework for high throughput metabolic testing that can be coupled with high throughput sequencing. It demonstrated the necessity for in-lab testing as non-functional genes can be annotated. I was able to identify pseudogenes in positive genotypes with negative phenotypes and identify novel proteins. This will lead to improved annotations and gene databases.