Different explanations exist on how HIV-1 subtype B spread in Central America, but the role of Guatemala, the Central American country with the highest number of people living with the virus, in this scenario is unknown. We investigated the evolutionary history and spatiotemporal dynamics of HIV-1 subtype B in Guatemala. A total of 1,047 HIV-1 subtype B pol sequences, from newly diagnosed ART-naïve, HIV-infected Guatemalan subjects enrolled between 2011 and 2013 were combined with published subtype B sequences from other Central American countries (n = 2,101) and with reference sequences representative of the BPANDEMIC and BCAR lineages from the United States (n = 465), France (n = 344) and the Caribbean (n = 238). Estimates of evolutionary, demographic, and phylogeographic parameters were obtained from sequence data using maximum likelihood and Bayesian coalescent-based methods. The majority of Guatemalan sequences (98.9%) belonged to the BPANDEMIC clade, and 75.2% of these sequences branched within 10 monophyletic clades: four also included sequences from other Central American countries (BCAM-I to BCAM-IV) and six were mostly (>99%) composed by Guatemalan sequences (BGU clades). Most clades mainly comprised sequences from heterosexual individuals. Bayesian coalescent-based analyses suggested that BGU clades originated during the 1990s and 2000s, whereas BCAM clades originated between the late 1970s and mid 1980s. The major hub of dissemination of all BGU, and of BCAM-II, and BCAM-IV clades was traced to the Department of Guatemala, while the root location of BCAM-I and BCAM-III was traced to Honduras. Most Guatemalan clades experienced initial phases of exponential growth (0.23 and 3.6 year-1), followed by recent growth declines. Our observations suggest that the Guatemalan HIV-1 subtype B epidemic is driven by dissemination of multiple BPANDEMIC founder viral strains, some restricted to Guatemala and others widely disseminated in the Central American region, with Guatemala City identified as a major hub of viral dissemination. Our results also suggest the existence of different sub-epidemics within Guatemala for which different targeted prevention efforts might be needed.