Twenty Japanese mattresses with various periods of use were collected from 11 localities in Japan in 2014. Samples of the mattresses including the padding were removed at 5 collection points in each mattress. e samples were divided into 3 layers (upper, middle, and bottom layers), and the fauna and density of house-dust mites were examined. A total of 12,734 mites of 10 families were collected and 97.0% of them belonged to Pyroglyphidae. e total number of mites was estimated as 4,640 to 394,240 per mattress. A positive correlation was found between the number of dead and alive mites and the number of exuviae. e dominant species in Pyroglyphidae was Dermatophagoides pteronyssinus (Trouessart) and D. farinae Hughes, and the proportion of D. pteronyssinus ranged from 0 to 100% among the samples. Multiple samples collected within the same locality (Tokyo, Kanagawa) showed a wide variation in the proportion of D. pteronyssinus. e distribution of mites in the 3 layers di ered among the 5 sampling points. More mites were distributed in the middle layers at sampling points located at the edge of the mattresses. e proportion of Pyroglyphid mites distributed in the upper and bottom layers ranged from 15.6 to 94.3%.