Technical Abstract: During the 12-year sugarcane breeding cycle in Louisiana, exchange of parental varieties and their progeny occurs routinely across different breeding stations and geographic locations for crossing and field evaluation purposes. This paper reports the major steps involved in the construction of a sugarcane molecular identity database as an additional breeding tool for the sugarcane breeders to maintain their cultivars and advanced breeding lines. Sugarcane (Saccharum spp.) is a complex aneu-polyploidy plant that propagates asexually through vegetative cuttings. To ensure genetic identity, a total of 1,025 sugarcane clones were genotyped from 2005 to 2008. The total includes 811 (79.1%) Louisiana, 45 (4.4%) Florida, 39 (3.8%) Texas, 130 (12.7%) foreign, and eight (0.8%) consultants or seed companies’ clones. Microsatellite genotyping was done following a standardized high throughput protocol on a fluorescence-capillary electrophoresis detection platform. Genotyping files were processed using GeneMapper™ software to reveal electrophoregrams that were manually checked for the status of 144 distinctive DNA fragments potentially amplifiable from 21 SSR markers. The presence (defined as “A”) or absence (defined as “C”) of these DNA fragments for each sample was recorded in an Excel spreadsheet. These linear sequences of As or Cs in an affixed order were then converted into DNAMAN® files to represent the molecular identities of these sugarcane samples that were stored in a local molecular identity database. By using the multiple sequence alignment program of DNAMAN® software, several Louisiana sugarcane clones that were mislabeled as cultivars and being used as parents in the breeding program were properly identified when their molecular identities did not group with the ones from known cultivars. The molecular identity database has been updated annually by continuing to genotype newly assigned clones from the advanced sugarcane breeding stages. The database provides molecular descriptions for cultivar registration articles to protect sugarcane breeders’ rights, enables the breeders to correctly use desired clones in attempted crosses, and determine the paternity of cross progeny. For sugarcane growers, the database ensures that the desired cultivars are growing in their fields when mapping discrepancies occur.