Tracheal acariosis in Apis mellifera (honey bee) is caused by the endoparasitic mite Acarapis woodi and affects the respiratory system. This disease has received very little empirical attention in Chile. Its prevalence is unknown, as is whether its prevalence varies during different times of the year. The objective of the present study was to determine the frequency of A. woodi and to estimate whether there is variation in its expression among beekeeping seasons. A total of 1,408 hives were sampled between 2012 and 2016. The results showed that the average prevalence in the country was 10.1%, with prevalences of 12.2% and 5.3% in the productive season and nonproductive season, respectively. A higher prevalence of tracheal acariosis was detected in the productive season. This study was the first to examine the prevalence of this disease in Chile. These findings provide insight into the prevalence of the disease in the Chilean context and can help to advance decisions about sanitary beehive management in the future.