We discovered that sequences essential for replication origin function are frequently conserved in sensu stricto Saccharomyces species. Here we use analysis of phylogenetic conservation to identify replication origin sequences throughout the Saccharomyces cerevisiae genome at base pair resolution. Origin activity was confirmed for each of 228 predicted sites--representing 86% of apparent origin regions. This is the first study to determine the genome-wide location of replication origins at a resolution sufficient to identify the sequence elements bound by replication proteins. Our results demonstrate that phylogenetic conservation can be used to identify the origin sequences responsible for replicating a eukaryotic genome.