Cyanovirin-N (CV-N) is a novel protein with broad-spectrum antiviral activity. Its homologs constitute a protein family known as CVNH (Cyanovirin-N homology), which possess the evolutionarily conserved anti-HIV (Human immunodeficiency virus) domain. In this study, more details about the patchy organism distribution of CVNH domain were explored by reconstructing gene trees. Duplicated CVNH sequences were also identified in a wide range of species including Aspergillus niger, Neosartorya fischeri NRRL 181, Penicillium chrysogenum Wisconsin 54-1255, Neurospora crassa, Cyanothece sp. PCC, and Ceratopteris richardii. Besides these findings, both the mechanistic and mechanistic-empirical combination (MEC) models were used to analyze the adaptive evolution of amino acid sites in the CVNH domain. Our results showed that: (1) neither model reveals significant sites undergoing positive selection; (2) purifying selection has played a dominant role during CVNH evolution; and (3) the MEC model better fits the CVNH data set. Furthermore, the ancestral branch leading to Cyanothece sp. PCC 7822 and 7424 were examined using "branch-specific" and "branch-site" models. Six positively selected sites (34L, 63L, 13H, 76C, 78K, and 80I) were identified on the branch.