In the present study, three orange cultivars (Citrus sinensis L. Osbeck), including Trovita, Early-golden, and Hamlin, were selected to produce not from concentrate (NFC) orange juice and from concentrate (FC) orange juice by the commercial manufacturing processes. Eight pairs of primers to obtain targeted fragments of different lengths were designed based on the chloroplast maturase K (matK) and nicotinamide adenine dinucleotide dehydrogenase (ndhF) genes. Polymerase chain reaction (PCR) coupled with capillary electrophoresis (CE) was used to analyze the effects of key processing steps such as extraction, homogenization, pasteurization, concentration, and secondary pasteurization on DNA degradation in the chloroplast matK and ndhF genes in Trovita orange juice. By comparing the differences in DNA degradation between NFC and FC orange juice, we selected the characteristic differential fragment sets and established a PCR-CE method to discriminate between NFC and FC orange juice. The developed method was applicable to analyze NFC and FC orange juice within shelf life. It was equally applicable to discriminate between NFC and FC orange juice manufactured from Early Golden and Hamlin sweet oranges. Finally, this method was used to test commercial NFC orange juice. The results were not consistent with the label information. The PCR-CE method will provide technical support for the effective regulation of the juice industry.