DNA barcoding is an approach for rapid and accurate species identification using a standard DNA region. Ideal barcodes should have high level of universality in PCR and sequencing. Although the nuclear ribosomal DNA (nrDNA) internal transcribed spacers 2 (ITS2) was proposed as potential DNA barcode for seed plants, including gymnosperms, no suitable ITS2 primer pair for gymnosperms available shows high universality in either aspect. To obtain a highly universal ITS2 primer pair, we newly designed three forward primers for ITS2 based on sequences in the 5.8S gene greatly conserved among 55 genera of gymnosperms. Combined with previously published reverse primers of ITS, seven candidate primer combinations for ITS2 were assessed for their universality. A total of 56 species, representing 40 genera from all the 12 families of the eight orders of gymnosperms, were sampled in this study. Three ITS2 primer combinations, 5.8SR/ITS4, 5.8SRa/ITS4, and 5.8SF2/S3R, were excluded due to their low universality or because of yielding double bands in the PCR in the familylevel assessment. The remaining four primer combinations, GYM_5.8SF1/ITS4, GYM_5.8SF1/S3R, GYM_5.8SF2/ITS4, and S2F/S3R, showed high PCR universality (100%) on all 56 sampled species. Based on our overall assessment of PCR and sequencing universality, brightness of PCR bands, and sequence quality and coverage, we propose the primer combination GYM_5.8SF2/ITS4 as ITS2 barcode for gymnosperms because it was shown to be the most “universal”.