Simple sequence repeat markers derived from expressed sequence tags (ESTs) are referred to as eSSRs. To develop molecular markers for non-model plants in Rosaceae, we investigated the transferability of apple eSSRs across seven fruit trees, belonging to four genera and 11 species of the Rosaceae family, including peach, quince, pear, loquat, apricot, cherry, and plum. Of the 98 apple eSSRs tested, 86 successfully amplified PCR products in at least one of the fruit tree species. Five apple eSSRs produced amplicons in more than five fruit tree species, and were deemed as a widely transferable Rosaceae marker set. Frequency of transferability of apple eSSRs across all seven fruit trees of Rosaceae varied widely among genera and species, with an average transferability of 29.0%. Overall, apple eSSRs transferred more easily to peach and pear than to plum and loquat. Interestingly, apple eSSRs present in coding sequences (CDS) showed higher levels of transferability to other fruit trees than those present in noncoding or untranslated regions (UTRs). Interestingly, apple eSSRs present in 5’UTRs showed lower frequencies of transfer than those present in 3’UTRs. The latter finding suggested that 5’UTRs might have diverged more rapidly than 3’UTRs in Rosaceae.