Stress resistance is an important trait expected of lactic acid bacteria used in food manufacturing. Among the various sources of stress, high temperature is a key factor that interrupts bacterial growth. In this regards, constant efforts are made for the development of heat-resistant strains, but few studies were done accompanying genomic analysis to identify the causal factors of the resistance mechanisms. Furthermore, it is also thought that tolerance to multiple stresses are equally important. Herein, we isolated one Enterococcus faecium strain named BIOPOP-3 and completed a full-length genome sequence. Using this strain, a two-step adaptive laboratory evolution (ALE) method was applied to obtain a heat-resistant strain, BIOPOP-3 ALE. After sequencing the whole genome, we compared the two full-length sequences and identified one non-synonymous variant and four indel variants that could potentially confer heat resistance, which were technically validated by resequencing. We experimentally verified that the evolved strain was significantly enhanced in not only heat resistance but also acid and bile resistance. We demonstrated that the developed heat-resistant strain can be applied in animal feed manufacturing processes. The multi-stress-resistant BIOPOP-3 ALE strain developed in this study and the two-step ALE method are expected to be widely applied in industrial and academic fields. In addition, we expect that the identified variants which occurred specifically in heat-resistant strain will enhance molecular biological understanding and be broadly applied to the biological engineering field.