Polychlorinated biphenyls (PCBs) are harmful and persistent organic pollutants. The influence of chiral PCBs acts mainly on the different enantiomer fraction, bioaccumulation and even degradation in environmental media. Solvents and temperatures existed almost everywhere during the analysis of extraction, purification, concentration and detection, which was often underestimated in previous studies. In our study, the configuration stability of the chiral PCBs was examined from solvent and temperature aspects. Transformation phenomena for the analytic stereoisomer monomers of PCB45, PCB95, and PCB149 affected by temperature were observed. We demonstrated that higher inlet temperatures could increase the sensibility for the low-concentration environmental samples, resulting in isomerization of chiral PCBs. Real rice samples were used to verify our analysis method. Combined with density functional theory, we verified the mechanism of isomer conversion with various numbers and sites of the -Cl substituent. PCBs with tetra-ortho substituents (2, 2', 6, 6') were relatively stable and showed the highest rotational barriers (Ea) at approximate 240 kJ mol-1. Others with trio-ortho substituents (2, 2', 6/6') showed Ea from 170 to 190 kJ mol-1, whose enantiomeric fractions would be affected by temperature during the analysis process for environmental detection. The method we developed was a promising means to understand the mechanism of isomerization and to predict stabilities of chiral PCBs.