Since the initial detection in 2003, Indonesia has reported 200 human cases of highly pathogenic avian influenza H5N1 (HPAI H5N1), associated with an exceptionally high case fatality rate (84%) compared to other geographical regions affected by other genetic clades of the virus. However, there is limited information on the genetic diversity of HPAI H5N1 viruses, especially those isolated from humans in Indonesia. In this study, the genetic and antigenic characteristics of 35 HPAI H5N1 viruses isolated from humans were analyzed. Full genome sequences were analyzed for the presence of substitutions in the receptor binding site, and polymerase complex, as markers for virulence or human adaptation, as well as antiviral drug resistance substitutions. Only a few substitutions associated with human adaptation were observed, a remarkably low prevalence of the human adaptive substitution PB2-E627K, which is common during human infection with other H5N1 clades and a known virulence marker for avian influenza viruses during human infections. In addition, the antigenic profile of these Indonesian HPAI H5N1 viruses was determined using serological analysis and antigenic cartography. Antigenic characterization showed two distinct antigenic clusters, as observed previously for avian isolates. These two antigenic clusters were not clearly associated with time of virus isolation. This study provides better insight in genetic diversity of H5N1 viruses during human infection and the presence of human adaptive markers. These findings highlight the importance of evaluating virus genetics for HPAI H5N1 viruses to estimate the risk to human health and the need for increased efforts to monitor the evolution of H5N1 viruses across Indonesia.