Phytoplasma is one of the most economically important pathogens, causing significant yield losses in many crops worldwide. During a survey in Bengaluru rural and Chikkabalpura areas of Karnataka, India, a total of 33 symptomatic plant samples including ornamentals, amaranthus, french bean, and sunhemp displaying phytoplasma symptoms were collected and confirmed as infected with phytoplasma through PCR targeting the 16S rRNA and secY genes. Pairwise sequence comparison and phylogenetic analysis of 16S rRNA and secY gene sequences confirmed the presence of ‘Candidatus Phytoplasma australasiaticum’ in antirrhinum, chrysanthemum, china aster, dianthus, amaranthus, french bean, and sunhemp. In-silico restriction fragment length polymorphism (RFLP) analysis indicated that phytoplasma strains infecting Antirrhinum, China aster, Dianthus, French bean, and Sunhemp represented distinct variants within the 16SrII phytoplasma group, with a threshold similarity coefficient of less than 0.98. A loop-mediated isothermal amplification (LAMP) assay was developed for detecting phytoplasma strains infecting the four ornamentals and other studied crops, targeting the phytoplasma 16S rRNA gene sequence with detection in 60 min. The comparative testing of LAMP products using various nucleic acid dyes and a colorimetric dye (hydroxy naphthol blue) confirmed the presence of phytoplasmas in symptomatic samples. This LAMP assay offers superior ease of use, cost-effectiveness, and rapid results for detecting phytoplasmas in symptomatic plants.