SUMMARY: GBS-SNP-CROP is a bioinformatics pipeline originally developed to support the cost-effective genome-wide characterization of plant genetic resources through paired-end genotyping-by-sequencing (GBS), particularly in the absence of a reference genome. Since its 2016 release, the pipeline's functionality has greatly expanded, its computational efficiency has improved, and its applicability to a broad set of genomic studies for both plants and animals has been demonstrated. This note details the suite of improvements to date, as realized in GBS-SNP-CROP v.4.0, with specific attention paid to a new integrated metric that facilitates reliable variant identification despite the complications of homologs. Using the new de novo GBS read simulator GBS-Pacecar, also introduced in this note, results show an improvement in overall pipeline accuracy from 66% (v.1.0) to 84% (v.4.0), with a time saving of ∼70%. Both GBS-SNP-CROP versions significantly outperform TASSEL-UNEAK; and v.4.0 resolves the issue of non-overlapping variant calls observed between UNEAK and v.1.0. AVAILABILITY AND IMPLEMENTATION: GBS-SNP-CROP source code and user manual are available at https://github.com/halelab/GBS-SNP-CROP. The GBS read simulator GBS-Pacecar is available at https://github.com/halelab/GBS-Pacecar. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.