Antisense long noncoding RNAs (ASlncRNAs) have been implicated in regulating gene expression in response to physiological cues. However, little is known about the evolutionary dynamics of ASlncRNA and what underlies the evolution of their expression. Here, using budding yeast Saccharomyces spp. and Naumovozyma castellii as models, we show that ASlncRNA repertoires have expanded since the loss of RNA interference (RNAi), in terms of their expression levels, their lengths and their degree of overlap with coding genes. Furthermore, we show that RNAi is inhibitory to ASlncRNA transcriptomes and that increased expression of ASlncRNAs in the presence of RNAi is deleterious to N. castellii, which has retained RNAi. Together, our results suggest that the loss of RNAi had substantial effects on the genome-wide increase in expression of ASlncRNAs during the evolution of budding yeasts.