Global efforts are underway to develop staple crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency, which is widespread among resource-poor farmers in the developing world. As a staple crop for more than 500 million people in sub-Saharan Africa, cassava (Manihot esculenta Crantz) enriched with provitamin A carotenoids could have immense nutritional impact. To this effort, a genome-wide association study (GWAS) was conducted with the aim of identifying genomic regions controlling variation for carotenoid concentration in cassava as a basis for undertaking systematic genetic improvement for carotenoid content. A high throughput genotyping-by-sequencing platform was used to genotype a diverse association panel of 591 genotypes and identified 179,310 single nucleotide polymorphisms (SNPs) distributed across the cassava genome. A genomic region stretching 1.37 Mb on chromosome 1 was identified with four SNPs significantly associated with carotenoid content in cassava. A survey of cassava genome sequence v6.1 positioned these SNPs in the vicinity of Manes.01G124200.1 locus, which is a known gene responsible for increase in accumulation of provitamin A carotenoids in cassava roots. This study provides one of the pioneer insights into the practical application of GWAS for dissecting the genetic basis of nutritional traits in cassava. Findings presented herein offer practical grounds for improving carotenoid content in cassava through marker-assisted breeding and genomic selection.