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Abstract Objective The aim of this study was to assess the magnitude, trend and reasons of rejection among referred specimens through referral network to the Amhara Public Health Institute (APHI) for laboratory testing. Results A total of 42,923 specimens were received at APHI reference laboratories. Of which, 221 (0.5%) specimens were rejected. CD4, HIV viral load, genexpert and EID specimens’ rejection rates were 0.7%, 0.6%, 0.3% and 0.2%, respectively. CD4 specimens were rejected due to wrong package (84.2%) and presence of clots (15.8%). Un-centrifuge (46.9%), hemolysis (19.8%) and use of wrong tube (17.7%) were the main rejection reasons for HIV viral load specimens. Although viral load specimen rejection was improved from 1.8 to 0% up to February/2018, the problem was reoccurred and continued to the end of May (1.3%) and June (0.3%) 2018. Moreover, CD4 specimen rejection (4.3%) was out of the established target in May, and exposed infant diagnosis (EID) specimen rejection became increased since March 2018. Hence, appropriate corrective and preventive actions and close follow up could reduce the problem of specimen referral network.