The eye lens of the Antarctic toothfish living in the -2 degrees C Southern Ocean is cold-stable. To investigate the molecular basis of this cold stability, we isolated, cloned and sequenced 22 full length crystallin cDNAs. We found two alpha crystallins (alphaA, alphaB), six beta crystallins (betaA1, betaA2, betaA4, betaB1, betaB2, betaB3) and 14 gamma crystallins (gammaN, gammaS1, gammaS2, gammaM1, gammaM3, gammaM4, gammaM5, gammaM7, gammaM8a, gammaM8b, gammaM8c, gammaM8d, gammaM8e, and gammaM9). Alignments of alpha, beta and gamma with other known crystallin sequences indicate that toothfish alpha and beta crystallins are relatively conserved orthologues of their vertebrate counterparts, but the toothfish and other fish gammaM crystallins form a distinct group that are not orthologous to mammalian gamma crystallins. A preliminary Fingerprinted Contig analysis of clones containing crystallin genes screened from a toothfish BAC library indicated alpha crystallin genes occurred in a single genomic region of ~266 kbp, beta crystallin genes in ~273 kbp, while the gamma crystallin gene family occurred in two separate regions of ~180 and ~296 kbp. In phylogenetic analysis, the gammaM isoforms of the ectothermic toothfish displayed a diversity not seen with endothermic mammalian gamma crystallins. Similar to other fishes, several toothfish gamma crystallins are methionine-rich (gammaM isoforms) which may have predisposed the toothfish lens to biochemically attenuate gamma crystallin hydrophobicity allowing for cold adaptation. In addition to high methionine content, conservation of alphabeta crystallins both in sequence and abundance suggests greater functional constraints relative to gamma crystallins. Conversely, reduced constraints upon gamma crystallins could have allowed for greater evolutionary plasticity resulting in increased polydispersity of gamma crystallins contributing to the cold-stability of the Antarctic toothfish lens.