Based on 2,256 common wheat reference varieties for DUS test in China, a DNA profile database was established to assist in distinctness test. We first analyzed the DNA profiles of 96 reference varieties using 81 fluorescently labeled SSR markers. A total of 731 alleles were detected with the number of alleles varying between 3 and 16 and the PIC values from 0.24 to 0.82. A DNA profile database consiting of 2256 wheat reference varieties was constructed using 42 fluorescently labeled SSR markers. A total of 148,493 datapoint were included with a missing rate of less than 5%. The genetic and genotypic datasets of 176 wheat varieties were collected and analyzed. The genetic similarities between the non-distinct candidate varieties and their most similar vairieties were above 90%. The candidate varieties with a genetic similarity of over 80% are assumed to be similar varieties, which are suggested to be further investigated for phenotypic variations in the field.