Areca catechu palm is an important cash plant in Hainan Island of China and even tropical regions worldwide. Areca catechu palm yellow leaf (AcYL) disease caused by the phytoplasmas is a devastating disease for the plant production. In the study, the phytoplasmas associated with the AcYL diseases were identified and characterized based on the conserved genes of the phytoplasmas, and genetic variation and phylogenetic relationship of the phytoplasma strains in the 16SrXXXII group was demonstrated. The results indicated that Areca catechu palm showing yellow leaf symptoms were single infected by ‘Candidatus Phytoplasma malaysianum’-related strains belonging to 16SrXXXII-D subgroup. BLAST and multiple sequence alignment analysis based on 16S rRNA and secA genes showed that the AcYL phytoplasmas shared 100% sequence identity and 100% homology with the ‘Ca. Phytoplasma malaysianum’-related strains. Phylogenetic analysis indicated that the AcYL phytoplasmas and ‘Ca. Phytoplasma malaysianum’-related strains belonging to 16SrXXXII group were clustered into one clade with a 100% bootstrap value. Based on computer-simulated digestions, 6 kinds of RFLP patterns within 16SrXXXII group were obtained and a novel subgroup in the 16Sr group was recommended to propose to describe the relevant strains in this 16Sr subgroup. To our knowledge, this is the first report that Areca catechu palm showing yellow leaf symptoms infected by ‘Ca. Phytoplasma malaysianum’-related strains belonging to 16SrXXXII group. And a novel 16Sr subgroup 16SrXXXII-F was proposed based on the systematical analysis of genetic variation of all the phytoplasmas within 16SrXXXII group. The findings of this study would support references for monitoring the epidemiology and developing effective prevention strategies of the AcYL diseases.