Background and objectives: One of the greatest challenges facing the healthcare of the aging population is frailty. There is growing scientific evidence that gait assessment using wearable sensors could be used for prefrailty and frailty screening. The purpose of this study was to examine the ability of a wearable sensor-based assessment of gait to discriminate between frailty levels (robust, prefrail, and frail). Materials and methods: 133 participants (≥60 years) were recruited and frailty was assessed using the Fried criteria. Gait was assessed using wireless inertial sensors attached by straps on the thighs, shins, and feet. Between-group differences in frailty were assessed using analysis of variance. Associations between frailty and gait parameters were assessed using multinomial logistic models with frailty as the dependent variable. We used receiver operating characteristic (ROC) curves to calculate the area under the curve (AUC) to estimate the predictive validity of each parameter. The cut-off values were calculated based on the Youden index. Results: Frailty was identified in 37 (28%) participants, prefrailty in 66 (50%), and no Fried criteria were found in 30 (23%) participants. Gait speed, stance phase time, swing phase time, stride time, double support time, and cadence were able to discriminate frailty from robust, and prefrail from robust. Stride time (AUC = 0.915), stance phase (AUC = 0.923), and cadence (AUC = 0.930) were the most sensitive parameters to separate frail or prefrail from robust. Other gait parameters, such as double support, had poor sensitivity. We determined the value of stride time (1.19 s), stance phase time (0.68 s), and cadence (101 steps/min) to identify individuals with prefrailty or frailty with sufficient sensitivity and specificity. Conclusions: The results of our study show that gait analysis using wearable sensors could discriminate between frailty levels. We were able to identify several gait indicators apart from gait speed that distinguish frail or prefrail from robust with sufficient sensitivity and specificity. If improved and adapted for everyday use, gait assessment technologies could contribute to frailty screening and monitoring.