Sorghum is an important crop widely used for food, feed, and fuel. Transcriptome-wide studies of 3' untranslated regions (3'UTR) using regular RNA-seq remain scarce in sorghum, while transcriptomes have been characterized extensively using Illumina short-read sequencing platforms for many sorghum varieties under various conditions or developmental contexts. 3'UTR is a critical regulatory component of genes, controlling the translation, transport, and stability of messenger RNAs. In the present study, we profiled the alternative 3'UTRs at the transcriptome level in three genetically related but phenotypically contrasting lines of sorghum: Rio, BTx406, and R9188. A total of 1,197 transcripts with alternative 3'UTRs were detected using RNA-seq data. Their categorization identified 612 high-confidence alternative 3'UTRs. Importantly, the high-confidence alternative 3'UTR genes significantly overlapped with the genesets that are associated with RNA N6-methyladenosine (m6A) modification, suggesting a clear indication between alternative 3'UTR and m6A methylation in sorghum. Moreover, taking advantage of sorghum genetics, we provided evidence of genotype specificity of alternative 3'UTR usage. In summary, our work exemplifies a transcriptome-wide profiling of alternative 3'UTRs using regular RNA-seq data in non-model crops and gains insights into alternative 3'UTRs and their genotype specificity.