Accurate and timely detection of transgene copy number in sugarcane is currently hampered by the requirement to use Southern blotting, needing relatively large amounts of genomic DNA and, therefore, the continued growth and maintenance of bulky plants in containment glasshouses. In addition, the sugarcane genome is both polyploid and aneuploid, complicating the identification of appropriate genes for use as references in the development of a high-throughput method. Using bioinformatic techniques followed by in vitro testing, two genes that appear to occur once per base genome of sugarcane were identified. Using these genes as reference genes, a high-throughput assay employing RT-qPCR was developed and tested using a group of sugarcane plants that contained unknown numbers of copies of the nptII gene encoding kanamycin resistance. Using this assay, transgene copy numbers from 3 to more than 50 were identified. In comparison, Southern blotting accurately identified the number of transgene copies for one line and by inference for another, but was not able to provide an accurate estimation for transgenic lines containing numerous copies of the nptII gene. Using the reference genes identified in this study, a high-throughput assay for the determination of transgene copy number was developed and tested for sugarcane. This method requires much less input DNA, can be performed much earlier in the production of transgenic sugarcane plants and allows much more efficient assessment of numerous potentially transgenic lines than Southern blotting.