Background: Guangdong, China is a critical epicenter for avian influenza virus (AIV) emergence, where H5N1 and highly pathogenic H7N9 were first identified. Monitoring AIV genotype distribution and genetic diversity in this region is essential for providing early warnings and mitigating the risk of human infections.Methods: In May 2025, a cross-sectional study was conducted across 4 representative cities in Guangdong. We collected 500 environmental swabs and 45 wastewater samples from 23 live poultry markets (LPMs). Samples were screened for influenza A virus (IAV) and subtyped for H5, H7, and H9. Next generation sequencing (NGS) and phylogenetic analyses were employed to characterize viral diversity and zoonotic potential.Results: AIV was detected in 49.2% (246/500) of environmental samples. H9 was the dominant subtype (38.2%), followed by H5 (3.2%) and H7 (0.8%); 2.4% of positive samples showed multi-subtype co-occurrence. “Non-restricted zone” markets (80.0%) and “waste storage areas” (68.4%) were identified as primary contamination hotspots. Phylogenetic analysis revealed that H5 and H9 viruses are accumulating mutations linked to enhanced human-type receptor affinity. Notably, H5 and H6 sequences exhibited long internal branches and clustered with isolates from other Asian countries rather than domestic strains, highlighting a significant gap in current domestic molecular surveillance and a lack of continuous evolutionary data for these genotypes.Conclusions: Widespread AIV contamination persists in Guangdong LPMs, dominated by H9 with low-level circulation of highly pathogenic subtypes. The identified surveillance gaps for H5/H6 and the presence of multi-subtype environments underscore the urgent need for strengthened, integrated molecular monitoring and stricter market regulation to mitigate public health risks.