Myostatin, which is also known as growth and differentiation factor 8 (GDF8), acts as a negative regulator of skeletal muscle growth. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in many animals including sheep. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to investigate MSTN in a diverse range of sheep breeds including the New Zealand (NZ) Romney, Coopworth, Corriedale, Dorper, Perendale, Suffolk, Merino, Dorset Down, Poll Dorset, Texel and other NZ cross-bred sheep. A total of 28 nucleotide substitutions were identified from nucleotide c.-1199 in the promoter region to c.*1813 (based on NCBI GenBank accession number DQ530260) and including the well-described substitution c.*1232G>A (MSTN g+6223G>A). Of these 28 substitutions, 3 were located in the promoter region, 3 in the 5'UTR, 11 in intron 1, 5 in intron 2 and 5 in the 3'UTR. One substitution in exon 1 (c.101G>A) potentially results in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. Ten of these substitutions have not been reported previously. The genetic variation revealed in this study suggests this gene is more variable than hitherto reported and provides a foundation for future research into how this variation affects muscle and growth traits.