Alternative splicing is considered a major mechanism for creating multicellular diversity from a limited repertoire of genes. Here, we performed the first study of genetic variation controlling alternative splicing patterns by comprehensively identifying quantitative trait loci affecting the differential expression of transcript isoforms in a large recombinant inbred population of Caenorhabditis elegans, using a new generation of whole-genome very-high-density oligonucleotide microarrays. Using 60 experimental lines, we were able to detect 435 genes with substantial heritable variation, of which 36% were regulated at a distance (in trans). Nonetheless, we find only a very small number of examples of heritable variation in alternative splicing (22 transcripts), and most of these genes colocalize with the associated genomic loci. Our findings suggest that the regulatory mechanism of alternative splicing in C. elegans is robust toward genetic variation at the genome-wide scale, which is in striking contrast to earlier observations in humans. Copyright © 2010 by the Genetics Society of America.