Abstract Background and Aims Fabry disease (FD), an X-linked lysosomal storage disorder caused by alpha-galactosidase A (α-Gal A) deficiency, leads to significant morbidity and early mortality due to progressive organ failure. Given the availability of enzyme replacement therapy, early identification of FD is crucial for improving patient outcomes. Method A multicenter screening was conducted among chronic hemodialysis patients across six centers. We utilized a two-tiered diagnostic approach. Initially, α-Gal A activity was measured from dried blood spots on filter paper. Subsequently, those with reduced enzyme levels underwent GLA gene mutation analysis. Results Out of 428 patients, 255 (102 females and 153 males) were selected for testing, leading to the analysis of 239 patients. In the male cohort, the average α-Gal A activity was 21.84 ± 11.9 µmol/L/h, with 34 males below the cut-off value (<15.3 µmol/L/h) but with normal lyso-Gb3 levels and no mutations. Among females, α-Gal A levels were typically within the normal range. Notably, one female was found to have a heterozygous variant of uncertain significance in the GLA gene [p.(Asp313Tyr)] in exon 6, with normal α-Gal A and lyso-Gb3 levels. Screening of other family members was not possible, as they resided abroad although we recommended it. Conclusion Screening for AFD in high-risk groups, such as hemodialysis patients, is challenging yet essential. Implementing α-Gal A measurement as a standard procedure for newly registered hemodialysis patients could prospectively identify potential Fabry carriers, offering more excellent utility than our cross-sectional study. While identifying index patients at a late disease stage may not benefit the patients themselves, it is crucial for early detection of family members, potentially altering their disease course. This proactive approach, supported by national and international guidelines, could significantly impact the management and outcomes of AFD.