Background: Sensitization to Blomia tropicalis is associated with asthma in various tropical and subtropical countries; however, information about the specific molecular components associated with this disease is scarce. Objective: Using molecular diagnosis, we sought to identify B tropicalis allergens associated with asthma in Colombia. Methods: Specific IgE (sIgE) to 8 B tropicalis recombinant allergens (Blo t 2, 5, 7, 8, 10, 12, 13, and 21) was determined using an in-house ELISA system in asthma patients (n=272) and controls (n=298) recruited in a national prevalence study performed in several Colombian cities (Barranquilla, Bogotá, Medellín, Cali, and San Andrés). The study sample included children and adults (mean [SD] age, 28 [17] years).Cross-reactivity between Blo t 5 and Blo t 21 was evaluated using ELISA-inhibition. Results: Sensitization to Blo t 21 (aOR, 1.9; 95%CI, 1.2-2.9) and Blo t 5 (aOR, 1.6; 95%CI, 1.1-2.5), but not Blo t 2, was associated with asthma. sIgE levels to Blo t 21 and to Blo t 5 were significantly higher in the disease group. Cross-reactivity between Blo t 21 and Blo t 5 is on average moderate in frequency; however, analysis of individual cases indicates that it may be very frequent (>50%) in some cases. Conclusions: Although Blo t 5 and Blo t 21 are considered common sensitizers, this is the first report of their association with asthma. Both components should be included in molecular panels for diagnosis of allergy in the tropics.