The transcription factorTbx5is expressed in the developing heart, eyes and anterior appendages. Mutations in humanTBX5cause Holt–Oram syndrome, a condition characterized by heart and upper limb malformations.Tbx5-knockout mouse embryos have severely impaired forelimb and heart morphogenesis from the earliest stages of their development. However, zebrafish embryos with compromisedtbx5function show a complete absence of pectoral fins, while heart development is disturbed at significantly later developmental stages and eye development remains to be thoroughly analysed. We identified a noveltbx5gene in zebrafish—tbx5b—that is co-expressed with its paralogue,tbx5a, in the developing eye and heart and hypothesized that functional redundancy could be occurring in these organs in embryos with impairedtbx5afunction. We have now investigated the consequences oftbx5aand/ortbx5bdownregulation in zebrafish to reveal thattbx5genes have essential roles in the establishment of cardiac laterality, dorsoventral retina axis organization and pectoral fin development. Our data show that distinct relationships betweentbx5paralogues are required in a tissue-specific manner to ensure the proper morphogenesis of the three organs in which they are expressed. Furthermore, we uncover a novel role fortbx5genes in the establishment of correct heart asymmetry in zebrafish embryos.