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Simple Summary: Nucleopolyhedroviruses of caterpillars (Lepidoptera) produce occlusion bodies (OBs) that protect the virions within a protein matrix. This allows the virus to persist in the environment until ingested by a susceptible insect. The soil is an important reservoir of OBs that can be transported to plants for transmission to the host caterpillar. The Spodoptera frugiperda multiple nucleopolyhedrovirus is a lethal pathogen of the fall armyworm, a major pest of maize, rice, and sorghum. Analysis of 186 soil samples collected from maize fields in southern Mexico, Belize, and Guatemala revealed that almost 19% of samples had OBs present at low concentrations. Genetic analysis revealed the presence of genetic diversity in the soil OB populations. The presence of OBs was higher in maize fields with living crops and in specific types of soils. These findings suggest that the soil could be a valuable source of genetic diversity for the design of virus-based insecticides to control this important pest. The occlusion bodies (OBs) of lepidopteran nucleopolyhedroviruses can persist in soil for extended periods before being transported back on to the foliage for transmission to the host insect. A sensitive insect bioassay technique was used to detect OBs of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) in 186 soil samples collected from maize fields in the southern Mexican states of Chiapas, Tabasco, Campeche, Yucatán, and Quintana Roo, as well Belize and Guatemala. Overall, 35 (18.8%) samples proved positive for SfMNPV OBs. The frequency of OB-positive samples varied significantly among Mexican states and countries (p < 0.05). Between 1.7 and 4.4% of S. frugiperda larvae that consumed OB-positive samples died from polyhedrosis disease. Restriction endonuclease analysis using PstI and HindIII confirmed that the soil-derived isolates were strains of SfMNPV and that genetic diversity was evident among the isolates. The prevalence of OB-positive soil samples did not differ with altitude or extension (area) of the maize field, but it was significantly higher in fields with the presence of living maize plants compared to those containing dead plants or crop residues (p < 0.05). Georeferenced soil samples were used to identify soil types on digitized soil maps. Lithosol and Luvisol soils had a higher than average prevalence of OB-positive samples (42–45% positive) (p = 0.006), as did Andosol, Gleysol, and Vertisol soils (33–60% OB-positive), although the sample sizes were small (<5 samples) for the latter three soils. In contrast, Cambisol soils had a lower than average prevalence of OB-positive samples (5% positive). Bioassays on Acrisol, Fluvisol, Phaeozem, and Rendzina soils resulted in intermediate levels of OB-positive samples. We conclude that certain soil types may favor OB persistence and virus-mediated biological pest control. The soil is also likely to provide a valuable source of genetic diversity for the design of virus-based insecticides against this pest. [ABSTRACT FROM AUTHOR]