Chenopodium L. is a relatively under-studied genus that includes the cultivated seed crop quinoa (Chenopodium quinoa Willd.). Quinoa is an allotetraploid (2n=4x=36, AABB genomes) that is cultivated by subsistence farmers and commercial growers in the Andean regions of South America. Approximately 60% of a quinoa seed is starch, a glucose polymer that is an important carbohydrate energy source in the human diet. Seed starch is normally comprised of amylose and amylopectin in a 1:3 ratio, but starches with different amylose:amylopectin ratios have different properties and potential uses. The accumulation of the amylose fraction of starch is controlled by a single dominant gene in quinoa, GBSSI. We report the sequencing and characterization of the GBSSI gene in 18 accessions of Chenopodium, including Andean quinoa and the related Mesoamerican chenopod grain species, C. berlandieri subsp. nuttalliae Saff. Two distinct homoeologs (GBSSIa and GBSSIb) were identified in the tetraploid accessions, and 19 different alleles were identified, including three null mutants – one in an accession of quinoa and two in a waxy landrace of C. berlandieri subsp. nuttalliae, referred to as ‘H02’. Expression analysis of the null mutants revealed that GBSSIa and GBSSIb were both strongly expressed late in quinoa and C. berlandieri subsp. nuttalliae seed development. Starch phenotyping indicated that ‘H02’ produced no amylose, likely due to its having two mutated copies of GBSSI. GBSSI sequences were used to analyze the phylogenetic relationships between quinoa and other members of the Chenopodium genus. This study and the discovery of Chenopodium GBSSI null-mutants will assist in the development of new Chenopodium crops with novel starches.