Maize (Zea mays L.) is one of the most important and widely cultivated crops in Argentina. Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a common maize pest capable of causing significant yield losses and is most destructive in late-planted maize in subtropical regions, going through five to six generations per growing season. The Bacillus thuringiensis (Bt) trait Herculex I Insect Protection technology by Dow AgroSciences and Pioneer Hi-Bred (HX I, event DAS-Ø15Ø7-1), expressing Cry1F protein, was launched in the 2005–2006 season in Argentina and was widely adopted because of the high level of efficacy against S. frugiperda, as well as other pests such as Diatraea saccharalis (J.C. Fabricius). However, increased late-season plantings, limited adoption of refuge, and properties of S. frugiperda biology (high number of generations and migratory behavior) have led to high S. frugiperda exposure to Cry1F and resistance selection pressure. Field efficacy monitoring has been conducted throughout the main maize production areas in Argentina from 2009 to 2016. Laboratory monitoring has been conducted throughout the same areas from 2010 to 2015. Here, we describe changes in field efficacy of HX I and the results of laboratory-based susceptibility monitoring conducted using purified Cry1F protein. Increases in larval survival and crop damage were evident throughout the 2012–2016 period and spanned the majority of maize production areas in Argentina. Over the same period, random larval collections showed increasing survivorship on diet containing purified Cry1F protein. These field and laboratory studies confirmed that resistance to Cry1F has developed and is now widely distributed in S. frugiperda populations in Argentina.