Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (  = 36/111, 32.4%) or prolonged (5 to 20 days) (  = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.