Nk homeobox genes are important regulators of many different developmental processes including muscle, heart, central nervous system and sensory organ development. They are thought to have arisen as part of the ANTP megacluster, which also gave rise to Hox and ParaHox genes, and at least some NK genes remain tightly linked in all animals examined so far. The protostome–deuterostome ancestor probably contained a cluster of nine Nk genes: (Msx)–(Nk4/tinman)–(Nk3/bagpipe)–(Lbx/ladybird)–(Tlx/c15)–(Nk7)–(Nk6/hgtx)–(Nk1/slouch)–(Nk5/Hmx). Of these genes, only NKX2.6–NKX3.1, LBX1–TLX1 and LBX2–TLX2 remain tightly linked in humans. However, it is currently unclear whether this is unique to the human genome as we do not know which of these Nk genes are clustered in other vertebrates. This makes it difficult to assess whether the remaining linkages are due to selective pressures or because chance rearrangements have “missed” certain genes. In this paper, we identify all of the paralogs of these ancestrally clustered NK genes in several distinct vertebrates. We demonstrate that tight linkages of Lbx1–Tlx1, Lbx2–Tlx2 and Nkx3.1–Nkx2.6 have been widely maintained in both the ray-finned and lobe-finned fish lineages. Moreover, the recently duplicated Hmx2–Hmx3 genes are also tightly linked. Finally, we show that Lbx1–Tlx1 and Hmx2–Hmx3 are flanked by highly conserved noncoding elements, suggesting that shared regulatory regions may have resulted in evolutionary pressure to maintain these linkages. Consistent with this, these pairs of genes have overlapping expression domains. In contrast, Lbx2–Tlx2 and Nkx3.1–Nkx2.6, which do not seem to be coexpressed, are also not associated with conserved noncoding sequences, suggesting that an alternative mechanism may be responsible for the continued clustering of these genes.