The present paper introduces improvements of the conventional selective amplification of microsatellite polymorphic loci (SAMPL) technique, that exploit AT-rich microsatellite primers. Generally, AT/AAT microsatellites are frequent components of eukaryotic genomes, but their ubiquity and polymorphic information content (PIC) could not be exploited yet, because standard SAMPL conditions did not allow amplifications. Here we report (i) on the design of new versatile AT-rich microsatellite primers, that are combined with (ii) a modified SAMPL adapter primer (called EcoRI-Short), and (iii) special polymerase chain reaction (PCR) amplification regimes. The novel SAMPL procedure expands the range of useful microsatellite primers to AT-rich sequences and produces a high number of bands and a clear banding pattern, and detects polymorphisms in otherwise nonpolymorphic genomes of plants (Dioscorea alata, D. rotundata) and a fungus (Mycosphaerella fijiensis).