Purpose: This study aimed to identify the working conditions (working hours, overtime work, number of night shifts, number of holidays, and work intervals) associated with fatigue, based on the shift patterns, and determine their thresholds. Methods: From January to February 2020, a web-based questionnaire was sent to 4601 shift work nurses at 47 hospitals in Japan. The multivariate logistic analysis was conducted to predict high- and low-fatigue groups by working conditions, and receiver operating characteristic analysis was performed to clarify the high-fatigue thresholds by shift pattern. Results: A total of 386 shift work nurses participated in this study. The threshold (fatigue was 3.0 or higher) of the two-shift rotation was 9 hours 50 minutes for daily working hours during day shifts (Odds ratio [OR] = 1.57, p < .01), 17 hours 15 minutes for daily working hours during night shifts (OR = 1.20, p < .01), and 8.0 days for the number of night shifts (OR = 1.09, p = .02). The threshold of the three-shift rotation was 9 hours 45 minutes (OR = 1.59, p < .01), 2.9 days for the number of midnight shifts (OR = 1.53, p < .01), and 2.0 times for the interval between day-shift and night-shifts within 12 hours (OR = 1.39, p < .01). Conclusion Working hours and the number of night shifts are important for two-shift rotation, and working hours for the assignment of midnight shift are important for three-shift rotations. Nurse managers should manage shifts according to nurses’ shift patterns.