Background: Blomia tropicalis sensitization is associated with asthma in different tropical and sub-tropical 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 eight B. tropicalis recombinant allergens (Blo t 2/5/7/8/10/12/13 and 21) was determined using an in-house developed ELISA system in asthmatic patients (n=272) and control subjects (n=298) recruited in a national prevalence-study performed in Colombian cities (Barranquilla, Bogotá, Medellín, Cali and San Andrés). Sample study included children and adults (mean age: 28±SD 17 years old). Cross-reactivity between Blot 5 and Blo t 21 was evaluated by 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; however, individual analysis indicates that may be high (>50%) in some cases. Conclusions: Although Blo t 5 and Blo t 21 has been described as common sensitizers, this is the first report of their association with asthma. Both components should be included in molecular panels for allergy diagnosis in the tropics.