This study was performed to investigate the prevalence and to characterize the genetic diversity of Histomonas meleagridis isolates in chickens in southern Vietnam. A total of 194 chickens, randomly selected from 18 backyard and 18 commercial flocks, were screened for H. meleagridis infection using both macroscopic diagnosis and an 18S rRNA gene-based PCR method. Overall, 12.9% of birds, representing 19 flocks, showed gross lesions typical for histomonosis whereas 25.3% of the birds from 29 flocks were positive by PCR assay. Following initial diagnostic approaches, H. meleagridis-positive samples were further analyzed by sequencing three different genomic loci; the 18S rRNA, alpha-actinin1, and rpb1. Thirteen samples from 12 flocks were genetically identified as H. meleagridis, demonstrating a flock and sample prevalence of 33.3% and 6.7%, respectively. There was no significant difference in prevalence between different farm types, age groups, and seasonality. Genetic analysis demonstrated minor heterogeneity of Vietnamese isolates with 99% homology to H. meleagridis sequences from the database. This is the first survey of the prevalence and genetic characterization of H. meleagridis in chickens in Vietnam.