Abstract: As one of the key enzymes involved in lignin decomposition of forest litter, laccase plays an important role in the carbon cycling in forest ecosystem. By using TA cloning and sequencing, a comparative study was conducted on the basidiomycetous laccase gene diversity  in the O horizon (litter layer) and A horizon (surface soil layer, 0-20 cm) in two subtropical forests (a primeval evergreen deciduous broadleaved mixed forest and an artificial masson pine forest). For the same soil horizons, the basidiomycetous laccase gene diversity and richness were higher in the primeval forest than in the masson pine forest; for the same forest ecosystems, the basidiomycetous laccase gene diversity and richness in the primeval forest were slightly higher in O horizon than in A horizon, but those in the masson pine forest were apparently lower in O horizon than in A horizon. The two forest soils had the same dominant laccase gene-containing basidiomycetous populations, and most of the populations had high similarity of amino acid sequence to Mycena sp. or Pleurotus sp. belonging to Agaricales. Comparing with the A horizon in primeval forest and the O horizon in masson pine forest, the O horizon in primeval forest and the A horizon in masson pine forest had a relatively uniform distribution of basidiomycetous populations. The nucleotide sequence similarity of basidiomycetous laccase gene between the O and A horizons in the masson pine forest was higher than that in the primeval forest. This study showed that vegetation and soil horizon had significant effects on the basidiomycetous laccase gene diversity and community structure, and the discrepancies in the substrate availability for basidiomycetes and in the soil pH induced by the vegetation and soil horizon could be the driving forces.