Citrus yellow mosaic disease is of high economic importance to the citrus industry in India. The mosaic disease is caused by Citrus yellow mosaic virus (CYMV) is a plant para-retrovirus, belongs to the Genus Badnavirus and family Caulimoviridae, with a DNA genome of 7.5 kb in size. The incidence of the disease ranged from 10-70% in citrus nurseries, and can cause reduction in fruit and juice yield up to 77 and 10%, respectively. The acid lime plants showing mosaic symptoms on the leaves as consisting of irregularly scattered light and dark green regions on the lamina were collected from Tirupati. Electron microscopy showed the presence of virus particles having bacilliform structure with size of ~30 x 130 nm. The CYMV genome was characterized using five sets of specific primers and a region of ORF III of CYMV associated with acid lime was cloned and sequenced. Upon sequencing of ORF III region produced a total of 899 bases. The homology search using NCBI revealed the highest sequence identity with other Indian isolate of CYMV infecting rangpur lime. Phylogenetic analysis of badnaviruses indicated that CYMV was most closely related to Cacao swollen shoot virus. Furthermore, high level of sequence conservation (89-99%) was noticed in the ORF III in three isolates infecting others species of citrus. The molecular tools developed in the present study could be used for large scale indexing of citrus plants to identify mosaic free plants and quick screening of planting material even at seedling stage without appearance of symptoms.