Antisense transcription, or transcription on the opposite strand of the same genomic locus as another transcript, has been observed in many organisms, including yeast. Several antisense transcripts are known to be conserved across various species of yeast, and a few antisense transcripts are associated with functional regulation of the sense transcript. We detect antisense transcription from approximately 90% of protein-coding genes, and antisense transcription is generally associated with histone modifications indicative of a transcriptionally active state. The pattern of genome-wide antisense transcription in two species of budding yeast, Saccharomyces cerevisiae and S. paradoxus, is widely evolutionarily conserved: Antisense transcripts exhibit conserved expression levels and localization with respect to gene annotations. Regions of genes exhibiting conserved antisense transcription also show less sequence divergence than regions of genes without antisense transcription. These findings provide further support that widespread antisense transcription is functional in yeast, and expand the catalog of putative functional antisense transcripts to include nonpolyadenylated transcripts. Because antisense transcripts are less divergent in sequence than expected, they likely contain sequences important to their function.