The performance of wavelength division multiplexing (WDM) in radio over fiber (RoF) systems is found to be strongly influenced by nonlinearity characteristics in side the fiber. The effect of four wave mixing (FWM) as one of the influential factors in the WDM for RoF has been studied here using Optisystem and Matlab. From the results obtained, it is found that the FWM effects have become significant at high optical power levels and have become even more significant when the capacity of the optical transmission line is increased, which has been done by either increasing the channel bit rate, and decreasing the channel spacing, or by the combination of both process. It is found that when the channel spacing is 0.1 nm, 0.2 nm and 0.5 nm the FWM power is respectively, becomes about -59 dBm, -61 dBm and -79 dBm. This result confirms that the fiber nonlinearities play decisive role in the WDM for RoF system. The simulation results obtained here are in reasonable agreement as compared with other numerical simulation results obtained, elsewhere, using different simulation tools.