Garlic (Allium sativum L.) is a traditional crop that has long been valued worldwide as food, condiment and medicine. Morphology-based authentication can hardly distinguish one garlic cultivar from other cultivars; however, the molecular markers for the identification of different garlic germplasms are yet to be investigated. The present study aimed to establish a reliable method that can specifically recognise ‘Taicangbaisuan’, a unique landrace in Jiangsu Province, China. We collected nine prevalent accessions of Chinese garlic cultivars, determined the discriminative ability of four DNA fragments (matK, psbA-trnH, nad1 and alliinase) among these cultivars and reconstructed their genetic relationships using inter simple sequence repeat (ISSR) markers. Our results revealed that the nucleotide substitutions of matK, psbA-trnH and nad1 could just divide these cultivars into two groups, while variable sites of alliinase could specifically identify ‘Taicangbaisuan’ from the other eight cultivars. Genetic relationship analysis based on ISSR markers indicated that these nine cultivars grouped into two main clusters that showed high accordance with morphological and agricultural characteristics, suggesting they may have been domesticated from two different ancestors. A unique band of ‘Taicangbaisuan’ was found in the products of ISSR-811 primer and a specific ISSR-SCAR marker was developed successfully according to this unique allele to authenticate ‘Taicangbaisuan’ from related garlic cultivars. The authentication method proposed in this study will be useful in the protection of excellent garlic germplasms and could serve as a reference for the molecular identification of other crops.