The reproducibility of relative cerebral blood volume (rCBV) measurements among readers with different levels of experience is a concern. This study aimed to investigate the inter-reader reproducibility of rCBV measurement of glioblastomas using the hotspot method in dynamic susceptibility contrast perfusion magnetic resonance imaging (DSC-MRI) with various strategies. In this institutional review board-approved single-center study, 30 patients with glioblastoma were retrospectively evaluated with DSC-MRI at a 3.0 Tesla scanner. Three groups of reviewers, including neuroradiologists, general radiologists, and radiology residents, calculated the rCBV based on the number of regions of interest (ROIs) and reference areas. For statistical analysis of feature reproducibility, the intraclass correlation coefficient (ICC) and Bland-Altman plots were used. Analyses were made among individuals, reader groups, reader-group pooling, and a population that contained all of them. For individuals, the highest inter-reader reproducibility was observed between neuroradiologists [ICC: 0.527; 95% confidence interval (CI): 0.21-0.74] and between residents (ICC: 0.513; 95% CI: 0.20-0.73). There was poor reproducibility in the analyses of individuals with different levels of experience (ICC range: 0.296-0.335) and in reader-wise and group-wise pooling (ICC range: 0.296-0.335 and 0.397-0.427, respectively). However, an increase in ICC values was observed when five ROIs were used. In an analysis of all strategies, the ICC for the centrum semiovale was significantly higher than that for contralateral white matter (P < 0.001). The inter-reader reproducibility of rCBV measurement was poor to moderate regardless of whether it was calculated by neuroradiologists, general radiologists, or residents, which may indicate the need for automated methods. Choosing five ROIs and using the centrum semiovale as a reference area may increase reliability for all users.