In order to reveal variation and evolution of M genes of human avian H5N1 influenza strains, the M genes of human avian H5N1 strains in Guangdong were sequenced and the M genes of global strains were searched out from Internet. They were analyzed by DNAStar 5. 0 and their revolutionary speeds were studied by means of combining the epidemiological data. It was found that M1 genes of 53 H5N1 strains and M2 genes of 51 strains during 1997-2006 were homologously classified into two groups: the strains from Hong Kong during 1997 (G I) were one group and the strains from Hong Kong, Vietnam, Thailand, Indonesia, China mainland, Turkey, Iraq, Azerbaijan, Egypt during 2003-2006 (G II ) were the another group. There were 20 substitutions of amino acids in M1 gene of all strains (7.94%, 20/252), where there were 9 amino acids in strains during 2003-2006 differing from the strains in 1997, meanwhile there were 22 substitutions of amino acids in M2 gene of all strains (22.7%, 22/97), where there were 4 amino acids in strains during 2003-2006 differing from the strains in 1997. In the synonymous variation, Ks values in M1 were 26.8 x 10(-6)-42.6 x 10(-6) Nt/d, and Ka values 4.39 x 10(-6)-6.98 x 10(-6) Nt/d, where there was more rapid speed of synonymous substitution than that of replacement, which showed that there existed less human immunological pressure and negative selective pressure by biological test. Ks values in M2 were 13.1 x 10(-6)-23.4 x 10(-6) Nt/ d, and Ka values 9.1 x 10(-6)-16.2 x 10(-6) Nt/d; where the ratios of Ks to Ka was 1.0-1.6 times as there was the neutral selective pressure in TL-676-05 strain. There was an amino acid substitution of S224, N in M1 gene of strains during 2003-2006 and an increas in a glycoprotein domain NSS224-226. The secondary structure of M2 protein varied as the substitution of C50 F of eight strains from Indonesia in 2005. The strains G I did not reemerge after Hong Kong human avian H5N1 influenza event. An increase of a glycoprotein domain NSS224-226 in M1 protein during 2003-2006 might be related with virus pathogenicity. Human avian H5N1 influenza M gene evolved frequently in nature, which might have an impact on its capacity of human-to-human transmission.