Routing flexibility is a major contributor towards flexibility of a flexible job shop manufacturing system. This article focuses on a simulation-based experimental study on the effect of routing flexibility and sequencing rules on the performance of a stochastic flexible job shop manufacturing system with sequence-dependent setup times while considering dynamic arrival of job types. Six route flexibility levels and six sequencing rules are considered for detailed study. The performance of manufacturing system is evaluated in terms of flow time related and due date–related measures. Results reveal that routing flexibility and sequencing rules have significant impact on system performance, and the performance of a system can be increased by incorporating routing flexibility. Furthermore, the system performance starts deteriorating as the level of route flexibility is increased beyond a particular limit for a specified sequencing rule. The statistical analysis of the results indicates that when flexibility exists, earliest due date rule emerges as a best sequencing rule for maximum flow time, mean tardiness and maximum tardiness performance measures. Furthermore, smallest setup time rule is better than other sequencing rules for mean flow time and number of tardy jobs performance measures. Route flexibility level two provides best performance for all considered measures.