House dust mites have been shown to be the most important source of allergen in Thailand. The results of our initial mite survey were in conflict with previous data reported 20 years ago. To evaluate the current condition of mite fauna in Thailand, we conducted a larger scale survey of house dust mites in dusts from three different parts of the country. A total of 630 mattress dust samples was collected from various types of housings from central, northern and northeastern parts of Thailand. House dust mites were identified and enumerated by standard techniques and 388 dust samples were analysed further for group I antigen by a two-site monoclonal antibody enzyme-linked immunosorbent assay. House dust mites were identified in 555/630 (88.1%) of dust samples collected; 27.6% of these 555 samples contained over 100 mites per gram of fine dust. Dermatophagoides pteronyssinus was the most abundant species found with a mean count of 87.2 mites per gram of dust (range 1–1460). Group I antigen was detected in 380/388 (97.9%) samples analysed and 330 of which (86.8%) contained over 2 μg/gm of group I antigen. The highest density of mites was found in dusts from northern Thailand whereas dusts with highest amount of antigen were those collected from the northeastern part. Some degree of correlation between mite count and mite allergen was observed (r2= 0.33, P< 0.001). No significant difference in the mite count and mite allergen was found among various types of housings. House dust mites are the source of allergens ubiquitously found all over Thailand. Dust from several houses surveyed in this study had significantly high level of group I antigens.