The aim of the study was to investigate whether seasonal differences in house dust mite (HDM) allergen exposure influence the circadian peak expiratory flow (PEF) amplitude in asthmatic children. Asthmatic children (n = 25) with a solitary allergy to HDM were studied in spring and in autumn. All used inhaled corticosteroids (ICS) regularly. Six days after withdrawal of ICS, PEF amplitude (every 4 h during 24 h, highest-lowest/percentage of mean value) was assessed. HDM allergen (HDMA) in living rooms, bedrooms, and mattresses was collected. HDMA levels were not always highest in autumn. PEF amplitudes in spring and autumn did not correlate with HDMA levels in the same season. However, the seasonal difference in PEF amplitude (autumn value - spring value) correlated positively and significantly with the seasonal difference in HDMA exposure levels from the mattresses (rho = 0.34, p < 0.05). Multivariate analysis showed that the seasonal difference in HDMA exposure in the mattress was the single parameter explaining seasonal difference in PEF amplitudes by 21.0% (p = 0.02). Our cross-sectional study showed a higher PEF amplitude not to be significantly associated with higher HDMA exposure in mattresses in a group of HDM-allergic asthmatic children. However, the change in HDMA exposure over seasons contributed significantly to the change in PEF amplitude after withdrawal of ICS in HDM-allergic asthmatic children.