Based on an association with extracellular matrix remodelling, mitosis, proliferation and adipogenic differentiation, the glycosaminoglycan hyaluronan (HA) was assessed as a marker for intramuscular fat (IMF) development (marbling) in bovine loin muscle (longissimus dorsi, LD). Loin samples collected from the quartering site of feedlot-finished Wagyu–Angus and Jersey–Limousin steers were assayed for percentage IMF (IMF%) and HA after assignment of AUS-MEAT marbling scores. There was a moderate phenotypic correlation (r² = 0.69) between IMF% and marbling score but little variance was explained by HA concentration. Breed was not a significant factor in marbling score or IMF% but did influence the HA concentration of the LD, with Wagyu–Angus steers having 2-fold more HA than Jersey–Limousin steers at the same marbling score. The non-linear decline in fat-adjusted HA levels as marbling score increased suggests that HA concentration was associated with lean growth potential of the muscle rather than adipogenesis. Using a different experimental approach, differences in distribution and amount of HA could not be discerned in histological sections of LD from age-matched Wagyu–Hereford heifers allocated to a low (score 1) or medium (score 3) marbling score group. These findings were consistent with the absence of differences between the two groups for other indicators of fatness (IMF% and P8 fat depth), maturity and myofibre characteristics despite an increase in oxidative capacity of the muscle with age. The data support the conclusion that the concentration of HA in the LD alone was not predictive of development of intramuscular fat.