Background: In vertebrates, cranial sensory placodes give rise to neurosensory and endocrine structures, such as the olfactory epithelium, inner ear, and anterior pituitary. We report here the establishment of a transgenic mouse line that expresses Cre recombinase under the control of Six1-21, a major placodal enhancer of the homeobox gene Six1. Results: In the new Cre-expressing line, mSix1-21-NLSCre, the earliest Cre-mediated recombination was induced at embryonic day 8.5 in the region overlapping with the otic-epibranchial progenitor domain (OEPD), a transient, common precursor domain for the otic and epibranchial placodes. Recombination was later observed in the OEPD-derived structures (the entire inner ear and the VIIth–Xth cranial sensory ganglia), olfactory epithelium, anterior pituitary, pharyngeal ectoderm and pouches. Other Six1-positive structures, such as salivary/lacrimal glands and limb buds, were also positive for recombination. Moreover, comparison with another mouse line expressing Cre under the control of the sensory neuron enhancer, Six1-8, indicated that the continuous and complex expression pattern of Six1 during sensory organ formation is pieced together by separate enhancers. Conclusions: mSix1-21-NLSCre has several unique characteristics to make it suitable for analysis of cell lineage and gene function in sensory placodes as well as nonplacodal Six1-positive structures. Developmental Dynamics 247:250–261, 2018. © 2017 Wiley Periodicals, Inc.