Myzus persicae is a cosmopolitan aphid that is highly polyphagous and an important agricultural pest. The subspecies M. persicae nicotianae has been described for highly specialized phenotypes adapted to tobacco (Nicotiana tabacum). In Chile, the population of M. persicae nicotianae was originally composed of a single red genotype that did not possess insecticide resistance mutations. However, in the last decade, variation in the colour of tobacco aphids has been observed in the field. To determine whether this variation stems from the presence of new genotypes, sampling was carried out across the entire distribution of tobacco cultivation regions in Chile. The aphids collected were genotyped, and the frequency of kdr (L1014F), super-kdr (M918T), modification of acetylcholinesterase (MACE) and nicotinic acetylcholine receptor β subunit (nAChRβ) mutations associated with insecticide resistance was determined. A total of 16 new genotypes of M. persicae nicotianae were detected in Chile: four of them possessed the MACE mutation, and none of them possessed the kdr, super-kdr or nAChRβ mutation. The previously described red genotype was not detected in any of the sampled fields over two seasons. These results raise questions about the mechanisms underlying changes in the genetic structure of M. persicae nicotianae populations in Chile. Future research aimed at addressing these questions could provide new insight into aphid evolution and agricultural practices.