Phylogenetic relationships among New World and eastern Atlantic species in the belonid genera Strongylura and Tylosurus were hypothesized using 3689bp of nucleotide sequence; including the entire mitochondrial (mtDNA) ATP synthase 6 and 8 genes; partial cytochrome b; 12S and 16S ribosomal genes; and introns and exons, 2 and 3 of the nuclear-encoded creatine kinase B gene. Concordant mtDNA and nuclear genealogies permitted well-supported inference of species relationships within Strongylura and Tylosurus, and of the chronology of diversification in the two genera. Our phylogenetic hypothesis permitted an assessment of Rosen's [Syst. Zool. 24 (1975) 431] model of species diversification across the eastern Atlantic to eastern Pacific marine biogeographic track. The spatial predictions of the Rosen model were generally supported, but not the temporal predictions. Furthermore, long branches leading to terminal Belonidae indicated that many species have persisted for millions of years or that nucleotide substitution rates were elevated for some clades. Though heterogeneity of nucleotide substitution rate was indicated across some belonid lineages, molecular clock estimates were used to hypothesize biogeographic scenarios for Strongylura across the eastern Pacific and Atlantic region. Furthermore, use of a molecular clock indicated; that early diversification among contemporary Strongylura may have been initiated by changes in Atlantic Ocean circulation precipitated by closure of the Tethys Sea; and provided approximate dates for the isolation of the freshwater species on the American continents.