The cranial neural crest cells (CNCCs) that give rise to the craniofacial skeleton are patterned by interactions between the dorsal-specifying jag1b pathway and the ventral-specifying edn1 pathway. In the zebrafish Danio rerio, wdr68 is essential for anterior dorsal and ventral jaw structures with a specific requirement for edn1 expression. The wdr68hi3812 retroviral insertion allele contains a 6kb provirus within the first exon, 131 nucleotides downstream of the start codon. Surprisingly, we found that the wdr68hi3812 allele is temperature sensitive (TS) with embryos reared at 24C yielding only modest joint fusions. In contrast, embryos raised at 32C present severe loss of the dorsal palatoquadrate (PQ) and ventral Meckel’s (M) cartilages. The transcription factors dlx1a/2a is expressed in a broad dorsal-to-ventral territory within the CNCC. At 24C, dlx1a/2a expression appears normal in wdr68 mutants, but at 32C wdr68 mutants display loss of anterior dlx1a/2a expression presaging the M and PQ defects. We further exploited the TS phenotype to define a developmental window during which wdr68 activity is required for M and PQ formation. We found this temporal window coincides with initiation of edn1 expression during late somitogenesis stages. We also developed a method for single-embryo genotyping and RT-PCR that readily detects wdr68 mRNA in wildtype sibling embryos but fails to detect wdr68 mRNA in mutant embryos at the prim-25 stage. Further analyses are underway to reveal the mechanism behind the TS nature of the wdr68hi3812 allele and examine the CNCC for potential defects in dorsal jag1b expression, migration, or survival. In summary, these findings refine our model for wdr68 activity in craniofacial development. Notably, the discovery that wdr68hi3812 is TS suggests that re-screening the hundreds of other retroviral insertional mutant zebrafish lines will likely reveal additional TS mutants and potentially novel phenotypes.