not-yet-known not-yet-known not-yet-known unknown Identifying molecular sensitization profiles in different regions is key to improve allergy diagnosis and treatment. This multicentric retrospective study analysed the results of 1010 ImmunoCAP ISAC® test performed in pediatric patients from 14 Portuguese hospitals. Sensitization to house dust mite (HDM) molecular allergens was found in 63.9% of patients, mainly to Der p 1, Der p 2, and Der p 23. Among monosensitized individuals Der p 23 stood out, underscoring its clinical significance for allergen-specific immunotherapy (AIT). Urbanization and coastal residence were associated with a higher risk of HDM sensitization, particularly to Dermatophagoides species. In contrast, no regional differences were observed for Lepidoglyphus destructor and Blomia tropicalis, potentially due to climate change and migratory flows, which may be altering traditional allergen distributions. Pollen sensitization affected 56.9% of patients, with grass pollens—mainly Phl p 1 and Cyn d 1—being the most prevalent. Sensitization increased with age, with Phl p 1 emerging as the dominant allergen after early childhood. Interior regions, characterized by greater vegetation, showed a significantly higher prevalence of pollen sensitization. Particular regional trends that reflected the native flora were noted, such as elevated sensitivity to Ole e 1 in Trás-os-Montes and Cup a 1 in Coimbra. Sensitization to Bet v 1 and related PR-10 proteins was associated with food allergy, though inland patients showed a higher rate of primary Bet v 1 sensitization without increased food allergy risk. These results highlight the importance of integrating regional allergen profiles into clinical practice to enhance diagnostic accuracy and guide tailored interventions.