DNA fingerprinting is a fast and accurate method for cultivar identification, which can overcome the limitation of morphological traits. We used DNA fingerprinting to identify 72 lotus (*Nelumbo*) cultivars collected from the Resources Garden of the Yuanmingyuan Park in Beijing. We used 1,409 samples of *N. nucifera* and 58 samples of *N. lutea* as a genetic background reference. Fifteen out of 104 pairs of nucleus microsatellite primers (nSSR) and 2 out of 17 pairs of chloroplast microsatellite primers (cpSSR), for a total of 17 pairs of fluorescent primers, were selected as the barcode for fingerprint identification of the 72 lotus cultivars. For the 15 nSSR primers, 94 alleles were examined (with an average of 6.27). Out of the 94 alleles, 11 belong to *N. lutea*, 65 belong to *N. nucifera*, and the remaining 18 alleles could not be identified. The polymorphism information content (*PIC*) range between 0.3899 and 0.8023, with an average value of 0.5748. For the two pairs of cpSSR primers, 13 haplotypes were examined. Among them, 9 haplotypes belong to *N. nucifera* and 4 haplotypes belong to *N. lutea*. Results of identification of all 17 pairs of primer markers showed that 19 cultivars included genes from *N. lutea*, and 8 cultivars had female parents from *N. lutea*. There were 36 cultivars (using 12 pairs of primers) which had at least one unique genotype. With a minimum of 8 pairs of primers, 68 cultivars could be distinguished. Among the 72 cultivars, four cultivars in two groups could not be distinguished based on the whole set of 17 primers. Using the core primer combination method, we developed specific DNA fingerprints for each of the 68 lotus cultivars. Based on the above results, we recommended 15 pairs of primers including 13 pairs of nSSR and 2 pairs of cpSSR as the core barcode for lotus cultivar identification.