The orthologs have similar or even identical functions in different species, share the anological regulatory pathways, and play the close or even same role among species. Furthermore, the vast majority of the biological core functions were assumed to a considerable number of orthologous genes in organisms. Orthologs was the most reliable choices for functional annotation and analysis of genomic sequences, whose unique biological characteristics demonstrated that comparative genomics research based on orthologs will certainly provide clues for detecting the origin, expression, and loss of important functional genes during the biological evolution in different organisms. In this review, the fundamental characteristics of orthologous genes and the relationship between the orthologs and comparison genomics were recounted. The corresponding approaches and the current status in comparative genomic research based on the orthologs were summarized.