The diversity and composition of drift invertebrate assemblages were evaluated along a longitudinal gradient of an altitudinal stream in southeastern Brazil. The main goal of this study was to evaluate the influence of seasonality, stream order, and some abiotic factors on invertebrate drift and the use of drifting invertebrate assemblages to assess aquatic invertebrate diversity. Drift samples were collected over a 24 h period using nets (open area of 0.08 m2; mesh 0.250 mm), partially submerged (60%) in the water column. Taxonomic richness, Pielou evenness (J), Shannon–Wiener diversity (H′), and total density of drift invertebrate assemblages were used in unpaired t-tests, Kruskal–Wallis and stepwise multiple regression analysis. The results showed a high taxonomic richness of aquatic invertebrates, with 91 taxa found. Chironomidae and Ephemeroptera represented together c. 80% of the total density of drift organisms. The drift approach allowed the collection of new and rare taxa, besides the knowledge of pupae stage of several chironomid genera. Significant differences in the taxonomic richness and diversity of drift invertebrate assemblages were found between the rainy and dry periods, indicating a significant influence of seasonality. An increase in water flow and electrical conductivity were associated with the increase in the taxonomic richness and diversity in the rainy period. No significant differences were found among the other abiotic variables among the stream orders.