Alignment of transcripts and genome sequences yielded a set of alternatively spliced transcripts in four angiosperm genomes: three dicotyledon species Medicago truncatula (Medicago), Populus trichocarpa (poplar) and Arabidopsis thaliana (Arabidopsis), and the monocotyledon Oryzae sativa (rice). Intron retention was the predominant mode of alternative splicing (AS) in each species, consistent with previous reports for Arabidopsis and rice. We analyzed the structure of 5'-splice junctions and observed commonalities between species. There was dependency of base composition between sites flanking the 5'-splice junction, with the potential to create a subset of splice sites that interact more weakly or strongly than average with U1 snRNA. Such altered nucleotide composition was correlated with splicing fidelity in all four species. For Medicago, poplar and Arabidopsis, but not in rice, alternative splicing was most prevalent for introns with decreased UA content, consistent with lower UA content for monocot introns and potentially reflecting evolved differences in splicing mechanisms. Similarly, the occurrence of AS between transcript Gene Ontology categories was positively correlated between Arabidopsis and Medicago, with no correlation between dicots and rice. Analysis of within-species paralogs and between-species reciprocal best-hit homologs yielded rare cases of potentially conserved AS events. Reverse transcriptase PCR and amplicon sequencing were used to confirm a subset of the in silico-predicted AS events within Medicago, as well as to characterize conserved AS events between Medicago and Arabidopsis.