Seasonal variability of macroinvertebrate assemblages was examined in two regions of South Africa: Western Cape and Mpumalanga. Sampling was undertaken at reference sites using the rapid bioassessment method SASS4 (South African Scoring System, Version 4). This study examined the influence of sampling season on the occurrence of taxa, macroinvertebrate assemblages and SASS scores. In the Western Cape a few taxa were more common at certain times of the year, and multivariate analysis of macroinvertebrate assemblages showed that assemblages grouped by season. The number of taxa and the Average Score per Taxon (ASPT) were significantly different among seasons in the Western Cape, with fewer taxa recorded in winter compared to summer, and significantly higher ASPT values recorded in winter and spring than summer and autumn. Whereas more taxa were recorded in autumn than in spring, a higher proportion of sensitive and high-scoring taxa were recorded in spring. In Mpumalanga macroinvertebrate assemblages did not group by season, and differences in SASS scores were not significant, although more taxa were recorded in winter than in spring or autumn, and ASPTs were slightly higher in winter than in spring. Results are discussed in relation to ecological reference conditions and the interpretation of bioassessment data.