BACKGROUND: Few studies have explored the role of ambient temperature in asthma exacerbation. OBJECTIVE: We aimed to explore the association of temperature with diurnal peak expiratory flow (PEF) variation and asthma exacerbation. METHOD: We developed a longitudinal study among asthmatic adults in 18 Chinese cities. Subjects recorded PEF in dynamic pulmonary function monitoring from 2017 to 2020. Linear mixed-effect model and generalized additive model with distributed non-linear models were used to assess the effect of temperature and temperature change between neighboring days (TCN) on diurnal PEF variation and the risk of asthma exacerbation. RESULT: We evaluated a total of 79,217 daily PEF monitoring records from 4467 adult asthmatic patients. There were significant increase of diurnal PEF variation and higher risk of asthma exacerbation with cold and sudden temperature drop. Compared with the referent temperature (99th percentile, 32 °C), exposure to moderate cold (25th percentile, 3 °C) and extreme cold (2.5th percentile, -7 °C) was associated with elevations of 1.28% and 1.16% in diurnal PEF variation over lag 0-2 days, respectively. The odds ratios of asthma exacerbation (determined by diurnal PEF variation >20%) at the two temperature cutoffs were 1.68 and 1.73. A sudden temperature drop (2.5th percentile of TCN, -5 °C) was associated with 1.13% elevation in diurnal PEF variation, and with increased risk of asthma exacerbation (odd ratio = 1.50) over lag 0-4 days. CONCLUSION: This large multicenter study provided the first-hand empirical evidence that cold temperature and a temperature drop may increase the risk of asthma exacerbation.