Abdominal lymphadopathy in Human Immunodeficiency Virus (HIV) infection remains a diagnostic challenge. We performed a prospective cohort study recruiting thirty-one symptomatic HIV+ patients with abdominal lymphadenopathy assessing diagnostic yield of endoscopic ultrasound (EUS) fine needle aspiration (FNA). Mean age was 38 years, 52% were female, mean CD4 count and viral load were 124 cells/pl, and 4 log respectively. EUS confirmed additional mediastinal nodes in 26 %. Porta-hepatis was the most common abdominal site. EUS FNA was subjected to cytology, culture and polymerase chain reaction (PCR) analysis. Mycobacterial infections were confirmed in 67.7% and 31% had reactive lymphadenopathy. Cytology and culture had low sensitivity whereas PCR identified 90% of mycobacterial infections. Combining appearance of EUS FNA and cytology a diagnostic algorithm was developed to indicate when analysis with PCR would be useful. PCR performed on an EUS guided aspirate was highly accurate in confirming mycobacterial disease and determining genotypic drug resistance.