The reemergence of malaria in the last decade in Madre de Dios, southern Peruvian Amazon basin, was accompanied by ecological, political, and socioeconomic changes related to the proliferation of illegal gold mining. We conducted a secondary analysis of passive malaria surveillance data reported by the health networks in Madre de Dios between 2001 and 2012. We calculated the number of cases of malaria by year, geographic location, intensity of illegal mining activities, and proximity of health facilities to the Peru–Brazil Interoceanic Highway. During 2001–2012, 203,773 febrile cases were identified in Madre de Dios, of which 30,811 (15.1%) were confirmed cases of malaria; all but 10 cases were due to Plasmodium vivax. Cases of malaria rose rapidly between 2004 and 2007, reached 4,469 cases in 2005, and then declined after 2010 to pre-2004 levels. Health facilities located in areas of intense illegal gold mining reported 30-fold more cases than those in non-mining areas (ratio = 31.54, 95% confidence interval [CI] = 19.28, 51.60). Finally, health facilities located > 1 km from the Interoceanic Highway reported significantly more cases than health facilities within this distance (ratio = 16.20, 95% CI = 8.25, 31.80). Transmission of malaria in Madre de Dios is unstable, geographically heterogeneous, and strongly associated with illegal gold mining. These findings highlight the importance of spatially oriented interventions to control malaria in Madre de Dios, as well as the need for research on malaria transmission in illegal gold mining camps.