This paper presents an evolutionary computing approach for the estimation of dynamic Origin-Destination (O-D) trip matrices from automatic traffic counts in urban networks. A multi-objective, simultaneous optimization problem is formulated to obtain a mutually consistent solution between the resulting O-D matrix and the path/link flow loading pattern. A genetically augmented microscopic simulation procedure is used to determine the path flow pattern between each O-D pair by estimating the set of turning proportions at each intersection. The proposed approach circumvents the restrictions associated with employing a user-optimal Dynamic Traffic Assignment (DTA) procedure and provides a stochastic global search of the optimal O-D trip and turning flow distributions. The application of the model into a real arterial street sub-network demonstrates its ability to provide results of satisfactory accuracy within fast computing speeds and, hence, its potential usefulness to support the deployment of dynamic urban traffic management systems.