To disclose mechanisms involved in temperature acclimation of fish muscle, eurythermal fish of medaka Oryzias latipes was subjected to cloning of myosin heavy chain genes (MYHs). We cloned cDNAs encoding fast skeletal muscle myosin heavy chain (MYH) isoforms from cDNA libraries of medaka acclimated to 10 and 30°C, and observed that different MYH cDNA clones are expressed in the two temperature-acclimated fish. Subsequently, we isolated several overlapping MYH contigs by shotgun cloning strategy from a medaka genomic library. Contig assembly of the complete medaka MYH (mMYH) locus of 219 kbp revealed a cluster of tandemly arrayed 11 mMYHs, in which eight genes are actually transcribed, with the rest three being pseudogenes. Expression analysis of the transcribed genes revealed that two genes were highly expressed each in medaka acclimated to 10 and 30°C whereas comparatively lower expression level of three genes were exclusively observed in medaka acclimated to 30oC. cDNAs of the remaining genes were too underrepresented in the libraries to determine the in expression levels and in the transcripts could only be obtained by reverse transcription-polymerase chain reaction. Deduced amino acid sequences in the loop 1 and loop 2 regions of mMYHs were highly variable suggesting that these isoforms were functionally different. The present findings consolidate our knowledge on teleost MYH multigene family and would provide further insight into the mechanisms by which expressions of individual MYH molecules are fine-tuned with environmental temperature fluctuations with further functional analysis of the genes concerned.