Viruses are the main pathogens responsible for acute upper respiratory tract infections; they spread easily among infants, young children, and the elderly population. The strong spreading capability of such viruses poses great difficulties for clinical diagnosis, treatment, and disease prevention and control. During the current coronavirus disease (COVID-19) pandemic, a clear understanding of the epidemiological characteristics of respiratory tract pathogens is essential for the timely identification of non-COVID-19 respiratory tract infections and enhancement of the efficiency of disease prevention and control. In this study, a total of 2,017 patients with upper respiratory tract infections in Kunming, Yunnan province of China, were included as subjects and divided into the Before COVID-19 and During COVID-19 groups. Differences in the infection rate of 13 common respiratory tract pathogens before and during the COVID-19 pandemic were determined using PCR in conjunction with capillary electrophoresis. Results indicated that the infection rate of respiratory tract pathogens (prevailing pathogens and infection status of different age groups) underwent significant changes after the start of the COVID-19 pandemic. Further in-depth research is required to improve the diagnosis, treatment, prevention, and control of respiratory tract infections.