The evolution and diversity of the supergroup Amoebozoa is complex and poorly understood. The supergroup encompasses predominantly amoeboid lineages characterized by extreme diversity in phenotype, behavior and genetics. The study of natural selection, a driving force of diversification, within and among species of Amoebozoa will play a crucial role in understanding the evolution of the supergroup. In this study, we searched for traces of natural selection based on a set of highly conserved protein-coding genes in a phylogenetic framework from a broad sampling of amoebozoans. Using these genes, we estimated substitution rates and inferred patterns of selective pressure in lineages and sites with various models. We also examined the effect of selective pressure on codon usage bias and potential correlations with observed biological traits and habitat. Results showed large heterogeneity of selection across lineages of Amoebozoa, indicating potential species-specific optimization of adaptation to their diverse ecological environment. Overall, lineages in Tubulinea had undergone stronger purifying selection with higher average substitution rates compared to Discosea and Evosea. Evidence of adaptive evolution was observed in some representative lineages and in a gene (Rpl7a) within Evosea, suggesting potential innovation and beneficial mutations in these lineages. Our results revealed that members of the fast-evolving lineages, Entamoeba and Cutosea, all underwent strong purifying selection but had distinct patterns of codon usage bias. For the first time, this study revealed an overall pattern of natural selection across the phylogeny of Amoebozoa and provided significant implications on their distinctive evolutionary processes.