The correspondence between physical classification schemes and the structure of biological assemblages has been intensively investigated in recent years for the implementation of bioassessment, biomonitoring and conservation programs. We evaluated the effect of temporal variability on the classification strengths of geographically and limnologically delimited regions and of lake connectivity for biological assemblages in a Neotropical floodplain. We sampled several biological groups from lakes in the Upper Paraná River floodplain over two years. Two classification criteria were used, considering (i) lakes associated with three sub-systems (regional scale classification) and (ii) lakes with and without watercourse connections to the river main channel (local scale classification). We directly evaluated the effects of sampling period, sub-systems and connectivity using Permutational Multivariate Analysis of Variance. For the significant factors, we calculated the extent to which the mean within-class similarity exceeded the mean between-class similarity (a measure of classification strength). Finally, we generated classes based on the biological data to evaluate whether they overlapped with the classifications we proposed. The correspondence between the classification criteria and the structures of the assemblages depended on the taxonomic group analyzed. A regional scale classification scheme based on sub-systems was important in accounting for the variability in the structures of different biological assemblages. Connectivity was important only in differentiating macrophyte and zooplankton assemblages. However, temporal variability is an important component affecting the responses of biological assemblages to physical divisions at different spatial scales. In addition, strengths of classifications were generally weak. The a posteriori classifications presented higher strengths of classifications, but also indicated that sub-systems, connectivity and sampling periods are important factors contributing to the variability of different biological assemblages. Further investigations in Neotropical floodplains should also take into account other local factors (e.g., aquatic macrophyte cover) to increase the predictive power of regionalization schemes.