BACKGROUND Patients with kidney disease on Hemodialysis (HD) are susceptible to Coronavirus Disease (COVID-19) due to multiple risk factors. OBJECTIVE This study aims to report the prevalence of antibodies against SARS-CoV-2 among patients on hemodialysis before vaccination in Brazil and to compare with clinical, demographic, and laboratory data. METHODS Blood samples from 398 Chronic Kidney Disease (CKD) patients treated in three different private institutions in Rio de Janeiro State, Brazil were submitted to the total anti-SARS-CoV-2 testing. Kidney, liver, and hematological markers were also determined. Respiratory samples were tested by real-time PCR for SARS-CoV-2 RNA and positive samples were subjected to high-throughput sequencing on the MinION device. RESULTS Overall, anti-SARS-CoV-2 prevalence was 54.5 % (217/398) and two individuals had SARS-CoV-2 RNA with variant B.1.1. High anti-SARS-CoV-2 seroprevalence was found in male gender and those with hospital admission in the last 3-months before the inclusion in the study. Lower red blood cell count was observed in the anti-SARS-CoV-2 seropositive group. High levels of anti-SARS-CoV-2 were found in those who reported symptoms, had low levels of eosinophils and low hematocrit, and who practiced physical activity. CONCLUSIONS High prevalence of anti-SARS-CoV-2 was found in CKD patients before the universal immunization in Brazil suggesting that dialysis patients were highly exposed to SARS-CoV-2.