Rapid Diagnostic Tests (RDTs) have been an important diagnostic tool for detecting P. falciparum malaria in resource-limited settings. Most tests are designed to detect the Histidine-rich Protein 2 (HRP2). Parasites lacking pfhrp2 and its homologous pfhrp3 have been reported in several regions, with prevalence reaching 100% in certain areas. To better characterize P. falciparum isolates circulating in the Brazil-Venezuela-Guyana tri-border region, we performed a comprehensive analysis of 365 samples collected between 2016 and 2018. Molecular and immunological methods were employed to detect HRP2 and confirm pfhrp2/3 deletions. Our findings point to a low prevalence (1%) of pfhrp2-deleted parasites confirmed by the lack of HRP2 detection. Among false-negative HRP2-RDT tests (6%), most were attributed to low parasite densities. A merozoite surface protein 2 (msp2)-based intra-host diversity analysis suggested overall low genetic diversity. The pattern of HRP2 sequences resembled that has been previously described in areas along the Brazil and French Guiana border. In conclusion, we have found a low prevalence of pfhrp2-deleted parasites in the north-central Guiana Shield, which contrasts with the findings reported at the Peru border. Continued surveys are necessary to monitor the prevalence of pfhrp2 deletion in this area characterized by a high number of cross-border malaria cases.