Аннотация:Abstract Twigs and seeds of Azadirachta indica (Neem) infected with dieback disease, collected from different regions of Karnataka, India were analysed to determine the pathogens. Phomopsis azadirachtae, the causal agent of dieback disease of neem was found to be seed borne. Phomopsis azadirachtae was isolated on PDA and MEA from dieback-infected neem twigs, seeds and embryo. Phomopsis genus-specific primers (5.8S r-DNA) were then used for the detection of Phomopsis azadirachtae, the causative agent of dieback of neem by polymerase chain reaction (PCR). Reactions were performed on DNA isolated from twigs, seeds and embryo of dieback-affected neem trees. Studies revealed the amplification of expected 141 bp DNA in Phomopsis azadirachtae isolated from various parts of diseased trees indicating the causal organism of dieback disease on neem. Isolation and identification by conventional technique takes around 15 – 21 days, whereas the present technique is capable of detecting very low propogules within 4 – 5 days.