A field survey was conducted in 2026 to investigate phytoplasma-associated diseases of chrysanthemum at ICAR-Indian Agricultural Research Institute (ICAR-IARI), New Delhi, India. During the survey, characteristic phytoplasma-like symptoms, including phyllody and little leaf, were observed in the chrysanthemum varieties Pusa Sentenary and Anmol, with disease incidence exceeding 11%. The present study aimed to identify and molecularly characterize the phytoplasma associated with these symptomatic chrysanthemum varieties. Total DNA extracted from symptomatic plants was subjected to nested PCR using phytoplasma 16S rRNA gene-specific primer pairs P1/ Tint and R16F2n/R16R2. The resulting amplicons were cloned, sequenced, and compared with reference phytoplasma 16S rRNA gene sequences available in the NCBI database. The 16S rRNA gene sequences obtained from chrysanthemum phytoplasma (CP) strains showed 99.92–100% nucleotide sequence identity with ‘Candidatus Phytoplasma’ australasiaticum related strains belonging to the 16SrII group reported from India and other countries. Phylogenetic analysis further revealed that the CP strains clustered closely with members of the peanut witches’ broom phytoplasma group (16SrII). In silico restriction fragment length polymorphism analysis of the 16S rRNA gene sequences generated a pattern corresponding to the 16SrII-D subgroup, with a similarity coefficient of 1.00. These results confirmed the association of ‘Candidatus Phytoplasma australasiaticum’ (16SrII-D) with phyllody and little leaf symptoms in both chrysanthemum varieties, Pusa Sentenary and Anmol. These findings highlight the potential significance of phytoplasma-associated diseases as an emerging and important constraint to chrysanthemum production in India, with possible implications for plant growth, flower quality, yield, and overall commercial value.