Extraocular muscle is fundamentally distinct from other skeletal muscle and demonstrates specific anatomical divisions, unique innervation, diverse myosin isoform expression patterns, a distinct genomic profile and differential involvement in neuromuscular disorders. The paired-like homeodomain transcription factor 2 (Pitx2) is known to regulate the formation of extraocular muscle development and in this report we show that its expression in adulthood also defines certain extraocular muscle traits. We found that expression of slow-MyHC and slow-tonic MyHC, along with contractile regulatory proteins troponin I and troponin T, is reduced during the first 3 weeks after birth in mice with conditional knockout of Pitx2, designated Pitx2(Δflox/Δflox). En grappe endplates, which are normally only found on slow-MyHC expressing fibres, were not identified in the Pitx2(Δflox/Δflox) extraocular muscle, suggesting that altered innervation was responsible for the loss in slow-MyHC expression. Extraocular muscle (EOM)-specific MyHC expressing fibres were dramatically reduced at P14 and rarely detected at 3 months in the Pitx2(Δflox/Δflox) mice. 2A-MyHC fibres, which are excluded from mid-belly region in wild-type mice, dominated the orbital layer with no apparent longitudinal variation in the Pitx2(Δflox/Δflox) mice. Pure 2X-MyHC fibres, only present at distal ends in the wild-type mice, populated the outer global layer in the mid-belly region of the Pitx2(Δflox/Δflox) mice. Pitx2 influences slow-MyHC, slow-tonic MyHC and EOM-MyHC expression in extraocular muscle and its absence leads to increased expression of 2X-MyHC and 2A-MyHC. Precise definition of the regulation of MyHC isoforms in extraocular muscle may allow their rational manipulation, in order to alter muscle contractility for therapeutic purposes.