72 Salvia miltiorrhiza lines from five different geographical populations located at Tai′an, Linyi, Laiwu, Heze and Weifang in Shandong province of China were studied by ISSR markers. The results indicated that 219 polymorphic bands were detected with 8 primers, average twenty-seven bands every primer. The percentage of polymorphic loci (P) was 98.63%, 81.28%, 66.67%, 66.21%, 51.14% and 50.68%, respectively at species level and Tai′an, Linyi, Laiwu, Heze and Weifang population level. The Nei′s gene diversity index and Shannon′s information index of species level were higher than population level. The Nei′s gene diversity index and Shannon’s information index of five populations were Tai′an ＞Linyi ＞Laiwu＞Heze＞ Weifang. Gene flow between the populations was 4.2352 based on genetic differentiation coefficient. UPGMA cluster analysis indicated that Laiwu population and Linyi population were the highest in Genetic identity which was the nearest in genetic relationships, and the genetic distance between Tai′an population and other four populations were the farthest. The results showed that Heze population and Tai′an population were relative independent populations, but there were still some gene exchange between five populations. Based on the study of population genetic structure, Tai′an population should be given a high priority consideration in Salvia miltiorrhiza populations in situ germplasm conservation strategies owing to its highest genetic diversity.