Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to absence of the mutations in poultry/environmental samples, suggested the mutations occurred during human infection and did not transmit further.