The objective of this study was to establish the extent to which lamb myofibre characteristics at ~8 months of age were influenced by sire (n = 9) estimated breeding values (EBVs) and pasture-based nutritional systems (low and high quality and availability, LOW and HIGH) from birth to slaughter (n = 56). Immunostaining of myosin heavy chain isoforms in longissimus muscle fibres revealed that as the post-weaning eye muscle depth EBV (PEMD) increased, the percentage of type 2B/X myofibres increased and the percentage of type 2A myofibres decreased. The percentage of type 2B/X myofibres also increased with increasing PEMD in the semimembranosus muscle, but not in the semitendinosus muscle. Post-weaning fat depth (PFAT) EBV was negatively related with the percentage of type 2B/X myofibres in longissimus and semitendinosus muscles and with the relative area of more glycolytic to more oxidative fibre types, although there was an interaction with nutritional system in the semitendinosus muscle, with this ratio increasing with the PFAT EBV among the LOW nutrition lambs. Overall, post-weaning weight (PWWT) EBV had less effect on myofibre characteristics than PEMD and PFAT EBVs. Average cross-sectional area of myofibres was more affected by the LOW than HIGH nutritional system in longissimus muscle (–25%) compared with semitendinosus (–21%) and semimembranosus (–15%) muscles. LOW nutrition resulted in a significantly smaller cross-sectional area of all myofibre types in longissimus muscle and of types 2A, 2AB/X, and 2B/X in semimembranosus and semitendinosus muscles. Overall, the relative area of glycolytic myofibres increased and/or more oxidative myofibre types decreased in LOW compared with HIGH nutritional system lambs in longissimus and semimembranosus, but not in semitendinosus muscle. The results demonstrate significant influences of sire EBVs on myofibre characteristics of lambs, the extent of which depends upon muscle type and nutritional regimen. More specifically, they suggest continued selection for increased muscling and reduced fatness in lambs are associated with shifts in the proportions of myofibre types, with potential for adverse effects on eating quality. They also show that the direction and magnitude of the effects of chronic, moderate postnatal nutritional restriction at pasture on myofibre characteristics vary between muscles with different contractile, metabolic, and/or functional characteristics.