Olive is an ancient oil-producing tree, widely cultivated in Mediterranean countries, and now spread to other areas of the world, including China. Recently, several molecular databases were constructed in different countries and platforms for olive identification using simple sequence repeats (SSRs) or single-nucleotide polymorphisms (SNPs). However, comparing their results across laboratories was difficult. Herein, hundreds of polymorphic single-copy nuclear sequence markers were developed from the olive genome. Using the advantage of multiplex PCR amplification and high-throughput sequencing, a fingerprint database was constructed for the majority of olives cultivated in China. We used 100 high-quality sequence loci and estimated the genetic diversity and structure among all these varieties. We found that compared with that based on SSRs, the constructed fingerprint database based on these 100 sequences or a few of them, could provide a reliable olive variety identification platform in China, with high discrimination among different varieties using the principle of BLAST algorithm. An example of such identification platform based on this study was displayed on the web for the olive database in China (http://olivedb.cn/jianding). After resolving redundant genotypes, we identified 126 olive varieties with distinct genotypes in China. These varieties could be divided into two clusters, and it was revealed that the grouping of the varieties has a certain relationship with their origin. Herein, it is concluded that these single-copy orthologous nuclear sequences could be used to construct a universal fingerprint database of olives across different laboratories and platforms inexpensively. Based on such a database, variety identification can be performed easily by any laboratory, which would further facilitate olive breeding and variety exchange globally.