TheMyoDgene was duplicated during the teleost whole genome duplication and, while a secondMyoDgene (MyoD2) was subsequently lost from the genomes of some lineages (including zebrafish), many fish lineages (includingAlcolapiaspecies) have retained bothMyoDparalogues. Here we reveal the expression patterns of the twoMyoDgenes inOreochromis(Alcolapia) alcalicausing in situ hybridisation. We report our analysis of MyoD1 and MyoD2 protein sequences from 54 teleost species, and show thatO. alcalica, along with some other teleosts, include a polyserine repeat between the amino terminal transactivation domains (TAD) and the cysteine-histidine rich region (H/C) in MyoD1. The evolutionary history ofMyoD1andMyoD2is compared to the presence of this polyserine region using phylogenetics, and its functional relevance is tested using overexpression in a heterologous system to investigate subcellular localisation, stability, and activity of MyoD proteins that include and do not include the polyserine region.