Huanglongbing (HLB or citrus greening or yellow shoot disease) is a devastating disease of citrus caused by nonculturable, fastidious phloem limited bacterium, Candidatus Liberibacter asiaticus and threatens the citrus industry in Asian countries. The putative causal agent of the disease is transmitted through insect vector or grafting with diseased budwood. The polymerase chain reaction (PCR) diagnosis is a more reliable and sensitive diagnostic tool for detecting greening bacterium than other conventional approaches like electron microscopy, DNA-DNA hybridization and immunofluorescence (IF) for detection of citrus greening. Results reveal that sodium sulphite method of DNA isolation provided higher yield and better quality DNA than other methods. To confirm the reliability of PCR, the greening bacterium was also detected in graft-inoculated plants, which showed typical greening symptoms. Results show amplification of 450 bp in PCR suggesting sampling in March is more suitable for PCR detection of greening bacterium. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a Candidatus Liberibacter asiaticus free nursery system.