H7N9 human influenza virus A/Anhui/1/2013 (Anhui2013) showed low pathogenicity in chickens, quail, and pigeons, with quail being the most susceptible among the species tested. IVPIE1-1, which was recovered from a dead chicken after intravenous inoculation of Anhui 2013, had broader tissue tropism in chickens than did the original inoculum, as well as amino acid substitutions in the polymerase acidic gene and neuraminidase gene segments, but its pathogenicity was not enhanced. Viruses obtained after passage of Anhui 2013 in 10- and 14-day-old embryonated eggs showed rapid accumulation of amino acid substitutions at the receptor-binding site of the hemagglutinin protein. Two strains obtained through egg passage, 10E4/14E17 and 10E4/10E13, replicated better in intranasally infected chickens than did the original Anhui 2013 strain, yet the new isolates showed low pathogenicity in chickens despite their amino acid substitutions. The increased virus replication in chickens of 10E4/14E17 and 10E4/10E13 was not correlated with temperature-sensitive replication, given that virus replication was suppressed at increased temperatures. The existence of highly susceptible hosts, such as quail, which permit asymptomatic infection, facilitates increased mutation of the virus through amino acid substitution at the receptor-binding site, and this might be one of the mechanisms underlying the prolonged circulation of H7N9 influenza virus.