Background Hepatitis C virus (HCV) infection is a chronic hepatotropic blood-borne infection. The transmission of HCV in patients undergoing hemodialysis (HD) is more common in comparison to the general population due to factors such as frequent blood transfusions, prolonged vascular access, and the potential for nosocomial infections. Western Rajasthan in India is home to numerous teaching and training hospitals that cater to a large number of HD patients. Understanding the seroprevalence and genotypic distribution of HCV in this specific patient population is crucial for assessing the extent of infection within this vulnerable group for targeted surveillance and developing effectively tailored treatment protocols in healthcare settings. Hence, this study was conducted with an aim to determine seroprevalence, seroconversion, and genotypes of HCV in HD patients at a tertiary care hospital. Methods This was a cross-sectional observational study. The duration of the study was from July 2019 to March 2022. In this study, the patients undergoing maintenance HD due to chronic kidney disease (CKD) were recruited. The data collected include patients' demographics, etiology of CKD, underlying other co-morbidities, duration of dialysis, and biochemical and blood count parameters. The patients recruited at the start of the study were screened for anti-HCV antibodies by HCV enzyme-linked immune sorbent assay (ELISA). The anti-HCV antibody-negative patients were followed up for the detection of anti-HCV antibodies. At the end of the follow-up period, all anti-HCV antibody negative samples in the pool of five and all anti-HCV antibody positive samples were subjected to a real-time polymerase chain reaction (RT-PCR) of 5' untranslated region (5'UTR) and core region, followed by line probe assay (LiPA). Results In this study, after applying inclusion and exclusion criteria, a total of 109 patients were recruited, out of which 64 (58.7%) were males and 45 (41.3%) were females. The age range of participants was 11-88 years with a mean and standard deviation of 46.75 and 16.35 years, respectively. A total of 39 patients (20 on screening, 19 on follow-up) were detected anti- HCV antibody positive. By RT-PCR, 24 patients tested HCV RNA positive (10 on screening, 14 on follow-up). Among 24 HCV RNA-positive samples, LiPA showed, HCV genotype 1a (n=21), genotype 3b (n=1), and two samples were detected to be inconclusive. Conclusion The increasing duration of dialysis is significantly associated with acquiring HCV infection. The majority of the cases of CKD in this geographical region are due to hypertensive nephropathy. There can be discordance between antibody and viral RNA positivity in HCV infection. The predominant HCV genotype identified in the dialysis ward of tertiary care hospital was genotype 1a.