Some tropical plant families, such as the Sapotaceae, have a complex taxonomy, which can be resolved using Next Generation Sequencing (NGS). For most groups however, methodological protocols are still missing. Here we identified 531 monocopy genes and 227 Short Tandem Repeats (STR) markers and tested them on Sapotaceae using target capture and NGS. The probes were designed using two genome skimming samples from Capurodendron delphinense and Bemangidia lowryi, both from the Tseboneae tribe, as well as the published Manilkara zapota transcriptome from the Sapotoideae tribe. We combined our probes with 261 additional ones previously published and designed for the entire angiosperm group. On a total of 792 low-copy genes, 638 showed no signs of paralogy and were used to build a phylogeny of the family with 231 individuals from all main lineages. A highly supported topology was obtained at high taxonomic ranks but also at the species level. This phylogeny revealed the existence of more than 20 putative new species. Single nucleotide polymorphisms (SNPs) extracted from the 638 genes were able to distinguish lineages within a species complex and to highlight geographical structuration. STR were recovered efficiently for the species used as reference (C. delphinense) but the recovery rate decreased dramatically with the phylogenetic distance to the focal species. Altogether, the new loci will help reaching a sound taxonomic understanding of the family Sapotaceae for which many circumscriptions and relationships are still debated, at the species, genus and tribe levels.