UniPrime is an open-source software (http://uniprime.batlab.eu), which automatically designs large sets of universal primers by simply inputting a gene ID reference. UniPrime automatically retrieves and aligns homologous sequences from GenBank, identifies regions of conservation within the alignment and generates suitable primers that can amplify variable genomic regions. UniPrime differs from previous automatic primer design programs in that all steps of primer design are automated, saved and are phylogenetically limited. We have experimentally verified the efficiency and success of this program by amplifying and sequencing four diverse genes (AOF2, EFEMP1, LRP6 and OAZ1) across multiple Orders of mammals. UniPrime is an experimentally validated, fully automated program that generates successful cross-species primers that take into account the biological aspects of the PCR.