Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.