N-nitrosamines, which are well-known pro-mutagens, are found in drugs, pickled food and tobacco. Therefore, controlling their concentrations is very important. When an HPLC, GC or NMR analysis is conducted to investigate certain asymmetrical N-nitrosamines, two sets of signals attributed to the asymmetric N-nitrosamine isomers are usually observed. However, few reports on the NMR assignment of asymmetrical N-nitrosamine isomers have been published. In this study, we investigated the NMR assignments of the Z/E isomers of six asymmetrical N-nitrosamines by means of density functional theory (DFT) calculations. The configuration of the major isomer of asymmetrical N-nitrosamine 3 was the Z-configuration. The configuration of the major isomers of asymmetrical N-nitrosamines 4–7 was the E-configuration. Then, we determined the Z/E ratios of these asymmetrical N-nitrosamines by means of variable temperature (VT) and room temperature (RT) 1H-NMR experiments. The ratios of the Z/E isomer 3 quickly increased beyond 100% in the VT 1H NMR experiments. The ratios of Z/E isomers 4–7 were increased in the range of 10–60% in the VT 1H NMR experiments. The results of this study indicate that identifying the isomers of asymmetrical N-nitrosamine is necessary to control the quality of N-nitrosamines for active pharmaceutical ingredients (APIs).