Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015-2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c, and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segment. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighboring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL-1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 102.5 EID50 /0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in Southeast Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.