Sorghum bicolor (L.) Moench is the fifth most valuable cereal crop globally. Although sorghum is tolerant to drought and elevated temperatures, it is susceptible to chilling, frost, and freezing stresses. Sorghum seeds planted in April may encounter frequent frost during late April and early May. Early spring freezing temperatures adversely affect crop development and yield. This study aims to identify genomic regions associated with frost tolerance at the seedlings stage. Breeding freeze-tolerant cultivars require selection for freeze tolerance in nurseries. However, the unpredictability of environmental conditions complicates the identification of freeze-tolerant genotypes. An indoor selection protocol has been developed to investigate the genetic determinism of freeze tolerance at the seedling stages and its correlation with several developmental traits. To accomplish this, we used two populations of recombinant inbred lines (RIL) developed from crosses between cold-tolerant (CT19, ICSV700) and cold-sensitive (TX430, M81E) parents. The derived RIL populations were evaluated for single nucleotide polymorphism (SNP) using genotype-by-sequencing (GBS) under controlled environments for their response to freezing stress. Linkage maps were constructed with 464 and 875 SNPs for the CT19 X TX430 (C1) and ICSV700 X M81E(C2) populations. Using quantitative trait loci (QTL) mapping, we identified six QTLs conferring tolerance to freezing temperatures. One QTL in the C1 population and four QTLs in the C2 population, explain 17.75-98% of the phenotypic variance of traits measured. Proline leaf content was increased in response to exposing the seedlings to low temperatures. Candidate QTLs identified in this study could be further exploited to develop frost-tolerant cultivars as proxies in marker-assisted breeding, genomic selection, and genetic engineering.