Next-generation sequencing (NGS) provides numerous tools for population and systematic studies. These tools are a boon to researchers working with non-model and poorly characterised organisms where little or no genomic resources exist. Several techniques have been developed to subsample the genomes of multiple individuals from related populations and species, so as to discover variable regions. We describe here the use of a modified AFLPseq method that provides a rapid and cost-effective approach to screening variable gene regions (SNPs) for multiple samples. Our method provides an adaptable toolkit for multiple downstream applications, which can be scaled up or down depending on the needs of the research question and budget. Using minor modifications to the protocol, we successfully recovered variable and useful markers that were applied to three case studies examining different scales of biological organisation, namely, from within populations to phylogenetic questions at the genus level and above. The case studies on Acacia and Eucalyptus generated genomic data across multiple taxonomic hierarchies, including demonstrating the detection of Acacia pinguifolia J.M.Black individuals used in restoration and their population origins, regional phylogeography of Acacia pycnantha Benth., and SNP-marker conservatism across some 70 million years of divergence among the Myrtaceae.