Beta diversity is the key and central issue of theoretical and applied ecological questions, including those regarding spatial variation along gradients and conservation biodiversity. In contrast to the alpha diversity of stream fish assemblages, little concern has been reached regarding the factor affecting of spatial variation in beta diversity along longitudinal gradients, which may be modified by scale, indices of beta diversity and pairwise methods. To understand the spatial patterns and processes in the species composition of stream fish assemblages along the river continuum, we need to disentangle the influence of different beta diversity indices and pairwise methods on the longitudinal patterns of beta diversity. In this study, we chose four beta diversity indices (Jaccard, Sørensen, Simpson and additive partitioning) and two pairwise methods (mean beta diversity between the focal assemblage and all other assemblages; beta diversity between focal and immediate neighbouring assemblages downstream) to quantify the spatial pattern of fish beta diversity along the river continuum. We found that (1) although the four diversity indices differed in emphasis, they showed the same pattern of spatial variation in the longitudinal gradients of the river; (2) the same diversity index using different pairwise methods calculation may result in completely different spatial patterns; (3) beta diversity indices and pairwise may not have a significant effect on the ecological application of beta diversity, at least in community assembly. In conclusion, the results of our study suggest that at least in the longitudinal gradient of the river there may not be much difference in quantifying the spatial variation of beta diversity; however, pairwise methods between sampling sites need to be chosen according to specific research objectives.