Analysis of 243 early Lyme disease patients reveals that males demonstrate 77% higher odds of positive two-tier serology compared to all females, with the disparity most pronounced against pre-menopausal women (2.93-fold higher odds). Post-menopausal women showed antibody responses similar to males, suggesting hormonal influences on immune recognition of Borrelia burgdorferi. This hormonal modulation of Lyme serology represents a significant clinical finding that could reshape diagnostic approaches. The traditional two-tier testing system may systematically underdiagnose Lyme disease in younger women, potentially leading to delayed treatment and increased risk of disseminated infection. Given that reproductive-age women comprise a substantial portion of tick-exposed populations through outdoor activities, this diagnostic blind spot could affect thousands annually. The research adds to growing evidence that sex hormones fundamentally alter pathogen recognition and antibody production. For clinicians, these findings suggest that negative serology in pre-menopausal women with characteristic erythema migrans rash should not rule out Lyme disease. The study's prospective design and focus on early-stage disease strengthens its clinical relevance, though replication across different geographic regions and tick species would validate broader applicability.