Natural isolates of the soil-dwelling bacterium Bacillus subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap-Phr regulatory systems. Most Rap-Phr systems have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap-Phr systems impact on bacterial differentiation. Here, we investigated each of the 12 Rap-Phr systems of B. subtilis NCIB 3610 for their role in biofilm formation. While Δ11 rap-phr mutants displayed increased matrix gene expression under biofilm inducing conditions, only some of the mutants demonstrated altered biofilm formation and colonization of Arabidopsis thaliana roots. Therefore, matrix gene expression does not directly correlate with biofilm formation in vitro and on the root. Our results suggest that each of the 12 Rap-Phr systems influences matrix gene expression, thereby allowing fine-tuning of the timing and level of matrix production in response to specific conditions, but additional factors also contribute to biofilm architecture and root colonization.