Purpose Serum galactomannan (GM) testing is widely used as a mycological marker in the diagnostic work-up of invasive aspergillosis (IA), but results near the assay cut-off may vary on repeat measurement. This study evaluated how often initially positive serum GM results remained positive when the same specimen was retested and how GM optical density index (ODI) values changed between the first and second tests. We also examined whether GM positivity and retest confirmation differed by sex, requesting service, underlying diagnoses, and initial GM-ODI ranges.Methods We retrospectively analyzed 27,014 serum samples from 3,121 patients requested by pediatric and adult services over seven years. Samples with an initial GM-ODI ≥ 0.5 and no known prior GM positivity were retested using a new aliquot of the same specimen within 72 hours.Results Of 27,014 serum samples, 1,918 were initially GM-positive, and 1,361 were retested within 72 hours. On repeat testing, 883/1,361 (65%) converted to negative, whereas 478/1,361 (35%) remained positive. GM-ODI values were significantly lower on retesting (p < 0.001). Final GM positivity did not differ by sex (p = 0.066) but was higher in pediatric than adult service requests (5.8% vs 3.4%, p < 0.001). Among final GM-positive specimens, the distribution of underlying diagnoses was associated with requesting service (p < 0.001). Retest positivity increased with higher initial GM-ODI ranges (p < 0.001) and varied across disease groups.Conclusion Repeat testing of initially GM-positive serum samples within 72 hours frequently resulted in reversion to negative. Confirmation was more likely at higher initial GM-ODI ranges. These findings indicate substantial short-term variability in serum GM results in routine practice and support cautious interpretation of low-positive results.