Alternative splicing alters gene expression in response to developmental and environmental cues. Splicing is an mRNA maturation process involving the removal of introns and the joining of exons through the recognition of splice-sites. Serine(S)/Arginine(R)-rich (SR) proteins are splicing factors which are thought to promote the use of splice-sites. SR proteins have been difficult to characterise due to lethality in animal systems and diversification in plant models making SR proteins functionally redundant. Here, we take advantage of the bryophyte Marchantia polymorpha, which harbors fewer SR proteins than most plants, to uncover the function and specificity of SR proteins. We show that single SR protein knockouts confer various viable phenotypes in M. polymorpha, including a loss of thermal response. We show that SR proteins have both unique target sites and targets shared across several SR proteins, defining SR protein specificity. And finally, we decipher specific motifs enriched among splice sites that are specifically affected in individual SR proteins, allowing us to infer specificity. Our results highlight the power of quantifying the usage of individual splice-sites in unravelling the specificity of distinct splicing regulatory proteins.