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Vertebrate pigmentation is a fundamentally important, multifaceted phenotype. Zebrafish,Danio rerio, has been a valuable model for understanding genetics and development of pigment pattern formation due to its genetic and experimental tractability, advantages that are shared across severalDaniospecies having a striking array of pigment patterns. Here, we use the sister speciesD. quaggaandD. kyathit, with stripes and spots, respectively, to understand how natural genetic variation impacts phenotypes at cellular and organismal levels. We first show thatD. quaggaandD. kyathitphenotypes resemble those of wild-typeD. rerioand several single locus mutants ofD. rerio, respectively, in a morphospace defined by pattern variation along dorsoventral and anteroposterior axes. We then identify differences in patterning at the cellular level betweenD. quaggaandD. kyathitby repeated daily imaging during pattern development and quantitative comparisons of adult phenotypes, revealing that patterns are similar initially but diverge ontogenetically. To assess the genetic architecture of these differences, we employ reduced-representation sequencing of second-generation hybrids. Despite the similarity ofD. quaggatoD. rerio, andD. kyathitto someD. reriomutants, our analyses reveal a complex genetic basis for differences betweenD. quaggaandD. kyathit, with several quantitative trait loci contributing to variation in overall pattern and cellular phenotypes, epistatic interactions between loci, and abundant segregating variation within species. Our findings provide a window into the evolutionary genetics of pattern-forming mechanisms inDanioand highlight the complexity of differences that can arise even between sister species. Further studies of natural genetic diversity underlying pattern variation inD. quaggaandD. kyathitshould provide insights complementary to those from zebrafish mutant phenotypes and more distant species comparisons.Author SummaryPigment patterns of fishes are diverse and function in a wide range of behaviors. Common pattern themes include stripes and spots, exemplified by the closely related minnowsDanio quaggaandD. kyathit, respectively. We show that these patterns arise late in development owing to alterations in the development and arrangements of pigment cells. In the closely related model organism zebrafish (D. rerio) single genes can switch the pattern from stripes to spots. Yet, we show that pattern differences betweenD. quaggaandD. kyathithave a more complex genetic basis, depending on multiple genes and interactions between these genes. Our findings illustrate the importance of characterizing naturally occuring genetic variants, in addition to laboratory induced mutations, for a more complete understanding of pigment pattern development and evolution.