The composition of the molecular species of triacylglycerols (TG) in subcutaneous fat biopsied from Japanese Black steers was studied during the fattening period. An analysis of the fatty acid (FA) composition of the TG showed that palmitic, palmitoleic, stearic and oleic acids were the major FA, together accounting for over 80% of the total FA. The concentrations of C14:0, C16:0 and C18:0 decreased between 10 and 30 months of age, while those of C16:1 c9 and C18:1 c9 increased during the fattening period. The major molecular species among more than 40 compounds were palmitoyl‐dioleoyl‐glycerol (POO) and dipalmitoyl‐oleoyl‐glycerol (PPO). POO showed the highest concentration during the fattening period. The levels of palmitoyl‐oleoyl‐linoleoyl glycerol and some tentatively identified molecular species increased during the fattening period, while the levels of tripalmitoyl, dipalmitoyl‐stearoyl‐glycerol, palmitoyl‐distearoyl and palmitoyl‐stearoyl‐oleoyl‐glycerol decreased with growth. A comparison of the experimental values of TG molecular species with theoretical values derived from the experimental FA contents showed that the FA distribution in TG was non‐random. TG synthesis favors the formation of TG molecular species containing at least one C16 FA, rather than three C18 FA.