© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Rho-associated protein kinases (ROCK) 1 and 2 are attractive drug targets for a range of neurologic disorders; however, a critical barrier to ROCK-based therapeutics is ambiguity over whether there are isoform-specific roles for ROCKs in neuronal structural plasticity. Here, we used a genetics approach to address this long-standing question by analyzing both male and female adult ROCK1 +/− and ROCK2 +/− mice compared to littermate controls. Individual pyramidal neurons in the medial prefrontal cortex (mPFC) were targeted for iontophoretic microinjection of fluorescent dye, followed by high-resolution confocal microscopy and neuronal 3D reconstructions for morphometry analysis. Increased apical and basal dendritic length and intersections were observed in ROCK1 +/− but not ROCK2 +/− mice. Although dendritic spine densities were comparable among genotypes, apical spine length was decreased in ROCK1 +/− but increased in ROCK2 +/− mice. Spine head and neck diameter were reduced similarly in ROCK1 +/− and ROCK2 +/− mice; however, certain spine morphologic subclasses were more affected than others in a genotype-dependent manner. Biochemical analyses of ROCK substrates in synaptic fractions revealed that phosphorylation of LIM kinase and cofilin were reduced in ROCK1 +/− and ROCK2 +/− mice, while phosphorylation of myosin light chain was decreased exclusively in ROCK1 +/− mice. Collectively, these observations implicate ROCK1 as a novel regulatory factor of neuronal dendritic structure and detail distinct and complementary roles of ROCKs in mPFC dendritic spine structure.