Interspecific hybridization plays an important role in plant adaptive evolution and speciation, and the process often results in phenotypic novelty. Hybrids can show changes in genome structure and gene expression compared with their parents including chromosomal rearrangments, changes in cytosine methylation, up- and downregulation of gene expression, and gene silencing. Alternative splicing (AS) is a fundamental aspect of the expression of many genes. However alternative splicing patterns have not been examined in multiple genes in an interspecific plant hybrid compared with its parents. Here we studied alternative splicing patterns in an interspecific Populus hybrid and its parents by assaying 40 genes using reverse transcription PCR. Most of the genes showed identical alternative splicing patterns between the parents and the hybrid. We found new alternative splicing variants present in the hybrid in two SR genes involved in the regulation of splicing and alternative splicing. The novel alternative splicing patterns included changes in donor and acceptor sites to create a new exon in one allele of PtRSZ22 in the hybrid and retention of an intron in both alleles of PtSR34a.1 in the hybrid, with effects on the function of the corresponding truncated proteins, if present. Our results suggest that novel alternative splicing patterns are present in a small percentage of genes in hybrids, but they could make a considerable impact on the expression of some genes. Changes in alternative splicing are likely to be an important component of the genetic changes that occur upon interspecific hybridization.