Alternative splicing alters gene expression in response to changes in developmental and environmental cues. Splicing is an mRNA maturation process, where regions of pre-mRNA between splice sites are removed, resulting in one or more unique mature mRNA splice-isoforms. Splicing factors, such as Serine(S)/Arginine(R)-rich (SR) proteins, bind to pre-mRNA and influence the utilisation of splice-sites. SR proteins are known to generally promote the usage of splice sites and are involved in several abiotic stress response pathways. However, little is currently known about how SR proteins influence splice-site choices. SR proteins are essential in animals, and highly redundant in plants, making their characterisation very difficult. Taking advantage of Marchantia polymorpha, which harbours fewer SR proteins, we show that single SR knockouts confer varied phenotypes, including loss of thermal response. More importantly, we are able to define SR protein specificity. Our results highlight the power of quantifying the usage of individual splice-sites in unravelling the specificity of distinct splicing regulatory proteins.