The aim of this study was to assess the effect of location within bovine longissimus dorsi (thoracis et lumborum) muscle (LTL) when determining glycogen concentration. Six locations in Angus heifers were sampled post mortem (EXP-1) and eight locations in live Charolais crossbred steers were biopsied (EXP-2). In EXP-1, there was more glycogen in the animals' left LTL (64.1±1.6 mmol/kg) versus the right LTL (57.0±1.6 mmol/kg) (P<0.05). Locations along the cranial-caudal axis within LTL did not differ in glycogen concentration. Results for EXP-2 did not confirm the sidedness effect. Instead, it showed that glycogen concentration was lower at the cranial sampling locations near the 10th rib (89±2.5 mmol/kg) than at the middle (97±2.0 mmol/kg) or caudal locations (96±1.9 mmol/kg) (P<0.005). Taking lactate accumulation into account (glycolytic potential) rendered those differences insignificant. Nevertheless, the tendency remained clear. The potential for dilution of glycogen by intramuscular fat deposits is discussed. The results indicate that one needs to be cautious in performing repeated sampling on bovine LTL.