A recent study (Bicer and Reiser, J. Muscle Res. Cell Motil., 25: 623-633, 2004) suggested considerable variation in the apparent molecular mass (M_a), deduced from electrophoretic mobility, in fast-type myosin light chains (MLCF), especially MLC1F, among mammalian species. Furthermore, there was an indication that MLC1F M_a generally correlates with species body mass, over an ~4,000-fold range in body mass. The results also suggested that M_a of other low molecular weight myofibrillar proteins is less variable and not as strongly correlated with body mass among the same species. The objective of this study was to test the hypotheses that the M_a of MLCs does, in fact, vary and correlate with species body mass. The electrophoretic mobilities of MLCF isoforms from nineteen species, varying in size ~500,000-fold were quantitated. The results confirm that the M_a of MLC1F and MLC2F vary significantly among mammals spanning a very broad range in body mass, the MLC1F M_a varies more than that of other low molecular weight myofibrillar proteins, and there is a significant correlation between species body mass and MLC1F M_a. Differences in MLC1F M_a among five species can be accounted for by differences in the reported amino acid sequence, especially the length of a common polyalanine region near the N-terminal actin-binding site. The possibility that the differences in MLC1F sequence among mammalian species, in and adjacent to the actin-binding region, are related to differences in modulation of cross-bridge kinetics in species with diverse locomotion kinetics is discussed.