In order to evaluate the performance of a newly developed time domain reflectometry (TDR) by measuring soil water content in the non-uniformly wetted soils, soil column was vertically divided into two layers in the laboratory, and the differences of soil water content between upper and lower layers were designed as 0.05, 0.10, and 0.15 cm3/cm3, and soil water content was measured under the conditions of different water content gradients, furthermore, the field test was also conducted to study the performance of the reflectometry in the non-uniformly wetted soils. Results showed that the new TDR could be used for measuring soil water content in the non-uniformly wetted soils and the root mean square error (RMSE) was less than 0.028 cm3/cm3. The RMSE increased as the differences of soil water content between upper layer and lower layer increased in the laboratory experiments. In field, when soil water content gradients of wet/dry and dry/wet were 0.011–0.026 and 0.026–0.064 cm3/ cm3, the RMSE was 0.037 and 0.027 cm3/cm3, respectively. The new TDR instrument can be used for measuring water content accurately in the non-uniformly wetted soils.