The objective of the study was to determine the effects of animal class and genotype of cattle on Muscularis longissimus thoracis et lumborum (LTL) nanostructure, ultimate pH (pHu), colour and tenderness of beef. We found significant positive relationships between distance travelled (DT) and meat temperature (Tm) (p<0.01); lairage duration (LDhr) and lightness of colour (L*) (p<0.01); ambient temperature (Ta) and L* (p<0.05) and LDhr and yellowness (b*) (p<0.05) of beef from Bonsmara cattle. Positive linear relationships were observed between DT and Tm (p<0.05) and DT and L* (p<0.01) of the non-descript cattle. There were no significant relationships between pre-slaughter stress and other beef quality parameters (pHu, Warner– Bratzler shear force [WBSF], redness [a*] and b*) of Bonsmara, Nguni and non-descript cattle. Muscle fibres differed among class and genotype and had an effect on meat quality. The Bonsmara, non-descript and Nguni cows and heifers had visible skeletal muscle fibres which were thin and long, promising improved tenderness of beef. Genotype and class had significant effects on meat quality parameters (Tm, pHu, L*, a*, b* and WBSF). The first important principal components as they appeared from the analysis were pHu, Tm, L*, a*, b* and WBSF. Therefore, animal class did not affect the nanostructure of beef; instead, meat tenderness was enhanced by the longer and visible muscle fibres. Nguni cattle produced meat of superior quality to that of the Bonsmara and the non-descript cattle.