Abstract Cytokinin is an influential hormone involved in numerous aspects of plant growth and development. A group of transcription factors–cytokinin response factors (CRFs) has been included as a side branch to cytokinin signaling pathway which also constitute a subset of the AP2/ERF family of transcription factor proteins. This study examined the expression patterns of two transcription factor genes SlCRF3 and SlCRF5 in tomato (Solanum lycopersicum) to determine their regulation in response to cytokinin and a variety of abiotic stress conditions. Analyses conducted during different developmental stages by RT-PCR or GUS reporter gene expression revealed that these genes are differentially expressed in vegetative and reproductive organs. qRT-PCR experiments were also performed to study regulation by the hormone cytokinin and abiotic stress conditions such as flooding, drought, osmotic, oxidative, and temperature. These showed that SlCRF3 and SlCRF5 have different patterns of regulation in leaf, stem, and roots with SlCRF5 showing greater induction in leaf or root tissue compared to SlCRF3 in most cases. Additionally, knockdown analysis for SlCRF5 revealed defects across development including leaf morphology, primary root growth, and lateral root formation. Together, these findings indicate that SlCRF3 and SlCRF5 are potential regulators of tomato developmental processes associated with cytokinin or abiotic stresses.