Phylogenetic systematics aims to describe the evolutionary history of all life and species’ interrelationships. One area of active study in molecular phylogenetics is phylogeography, which examines how genetic variation is structured across the geographic distribution of species. Within Gobiiformes, the genus Ctenogobius is a group of fifteen understudied species presently classified within Family Oxudercidae, subfamily Gobionellinae. The genus has undergone numerous taxonomic revisions. Formerly regarded as synonymous with the genus Gobionellus, Ctenogobius was resurrected by Pezold in 2004. However, since then, other work has suggested that species diversity within Ctenogobius is greater than currently described. Phylogeographic studies of other species of gobies with similar distribution and ecology as Ctenogobius spp. (Gobiosoma bosc, Eucyclogobius newberryi, Gnatholepis sp., etc.) have revealed evidence of high levels of cryptic diversity. For example, the darter goby Ctenogobius boleosoma has a large, disjunct distribution spread across the Atlantic coastline of the Americas, matching the distribution of the Naked Goby, Gobiosoma bosc. Due to its unique ecology and distribution, Ctenogobius boleosoma and its genus are perfect for studying both phylogeography and speciation. Using a phylogeny developed using ddRAD-seq data, I described the interrelationships of 10 Ctenogobius species using genetic data, focusing primarily on the darter goby C. boleosoma. I found support for a novel lineage within Ctenogobius that may be a cryptic species. Next, I then described the fine scale population structure of the species C. boleosoma for the first time ever, showing there is not much structure present beyond what was found in Chapter 1. Finally, I conclude with a comparative transcriptomic study looking at differential gene expression between two members of Ctenogobius – C. boleosoma and C. shufeldti – and describing 17 genes associated with ion channel transport proteins differentially expressed between the two species.