Mesp proteins play crucial roles in the formation of heart, vasculature and somites during vertebrate embryogenesis. We have used phylogenetic and genomic analysis, combined with qRT-PCR and in situ hybridization, to characterize two novel additional mesp genes in zebrafish, mesp-ab and mesp-bb, and describe their expression pattern in wild type and segmentation mutants. Both mesp-ab and mesp-bb are expressed in early mesoderm with mesp-ab expression starting during late blastula stages and mesp-bb expression initiating later, at the end of gastrulation. During somitogenesis, both mesp genes are expressed dynamically in the anterior presomitic mesoderm. mesp-ab is expressed in presumptive somites S-I and S-II, while mesp-bb is detected in S-I, S-II and S0, with expression restricted to the rostral compartment of presumptive somites. We show that the segmentation clock program regulates expression of these newly identified zebrafish mesp genes in a similar manner to their ohnologs, mesp-aa and mesp-ba. We also present evidence that zebrafish, minnow and salmon retained these additional mesp genes after the teleost whole genome duplication, while medaka, stickleback, fugu and tetraodon did not. Finally we show that although expression and regulation of zebrafish mesp genes appears highly comparable, there is no conservation in non-coding regions with other teleosts. In this study we have completed the description of the Mesp family in zebrafish, which will enable correct genome annotation and facilitate further functional studies on the role of these proteins in zebrafish.