ABSTRACT Whole-genome alignment allows researchers to understand the genomic structure and variations among the genomes. Approaches based on direct pairwise comparisons of DNA sequences require large computational capacities. As a consequence, pipelines combining tools for orthologous gene identification and synteny have been developed. In this manuscript, we present the latest functionalities implemented in NGSEP 4, to identify orthogroups and perform whole genome alignments. NGSEP implements functionalities for identification of clusters of homologus genes, synteny analysis and whole genome alignment, and visualization. Our results showed that the NGSEP algorithm for ortholog identification has competitive accuracy and better efficiency in comparison to commonly used tools. The implementation also includes a visualization of the whole genome alignment based on synteny of the orthogroups that were identified, and a reconstruction of the pangenome based on frequencies of the orthogroups among the genomes. Finally, our software includes a new graphical user interface. We expect that these new developments will be very useful for several studies in evolutionary biology and population genomics.