Objective. To describe the barriers identified by the health staff for the screening of human immunodeficiency virus (HIV) in indigenous populations at health centers located in four regions of the Peruvian Amazon. Materials and Methods. Mixed study. The qualitative phase was exploratory and the quantitative phase was based on a cross-sectional descriptive study of exploratory character. Eighty-seven (87) facilities were selected and a survey was applied. Additionally, 51 interviews were conducted to the health staff of some of the establishments. The interviews were codified by means of software Atlas.ti 7.0. For the surveys a descriptive statistical analysis was made. Results. The barriers identified were geographic, sociocultural, and others linked to the health system. It was more frequent to find shortage of inputs or reagents in health centers (40.0%) as compared to clinics and (25.0%) and hospitals (11.1%). The main aspect that limited the screening campaigns for indigenous population was the lack of budget (59.3%), the lack of human resources, people not accepting to have blood samples taken, as well as the lack of supplies or reagents for the diagnostic tests. Conclusions. The main barriers identified by the health staff are mostly those linked to the health system and those of a sociocultural nature. We recommend considering them for the improvement of the supply of the services, as well as for its cultural adjustment.