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In radio frequency identification (RFID) system, the reader-to-reader interference leads to reader collision that disturbs readers in tag recognition. The existing reader anti-collision techniques based on TDM or FDM are inefficient solutions to the reader collision problem since they do not consider jointly the distance between the interfering readers and the frequencies, and time slots that readers use. In this paper, we formulate a TDM/FDM-based reader-to-reader interference model to analyze the effects of the above factors and an optimization problem to maximize the RFID system performance. We then propose a novel resource allocation technique based on genetic algorithm (RA-GA) to solve the optimization problem. The RA-GA includes a unique encoding scheme and fitness function to obtain an optimal resource allocation for the RFID system effectively. Simulation results show that the RA-GA is superior to the resource allocation based on random method.