Maize (Zea mays L.) is one of the main staples in the Peruvian Andes. Phenotypic diversity is extremely high in terms of plant morphology, grain colour, grain size and uses. However, little is known about the genetic component of the Peruvian maize. Genotyping-by-sequencing was used, for the first time, to infer the genetic diversity and population structure of 25 accessions of maize collected from two ecoregions of the departments of the Peruvian highlands, Cajamarca and Huancavelica, to provide a basis for the conservation and breeding of this crop genetic resource. A total of 29,332 single-nucleotide polymorphisms were identified along all 10 chromosomes of maize. STRUCTURE analysis revealed two clusters (open-pollinated cultivars and landraces). Principal coordinate analysis and our dendrogram showed that these accessions of Peruvian maize do not group based on their geographic origin but on their improvement status, however, this is weakly supported. Average genetic diversity was very high (0.35). Analysis of molecular variance showed a reduced variation between populations (2.85%) and indicated that variability within populations is 97.15%. The lowest population divergence was zero for populations of Colcabamba versus Pichos and Daniel Hernandez versus Colcabamba and the highest population divergence (0.041) was observed for population Cajamarca versus Santiago de Tucuma. As expected, FIS values were positive. Additional samples from other Andean and west coast Peruvian localities are needed to provide a better understanding of the genetic components of this important crop aiming to develop a modern breeding programme of maize in Peru.