Complex traits and disease co-morbidity in humans and in model organisms are the result of naturally occurring polymorphisms that interact with each other and with the environment. To ensure the availability of the resources needed to investigate biomolecular networks and ultimately systems level phenotypes, we have initiated breeding of a new genetic reference population of mice, the Collaborative Cross. This population has been designed to optimally support systems genetics analysis. Its novel and important features include high levels of genetic diversity, a large population size to ensure sufficient power in high-dimensional studies, and high mapping precision through accumulation of independent recombination events. Implementation of the Collaborative Cross has been in progress at the Oak Ridge National Laboratory (ORNL) since May 2005. This is achieved through a software assisted breeding program with fully traceable lineages, performed in a uniform environment. Currently, there are 650 lines in production with almost 200 lines over seven generations of inbreeding. Retired breeders enter a high-throughput phenotyping protocol and DNA samples are banked for analysis of recombination history, allele loss, and population structure. Herein we present a progress report of the Collaborative Cross breeding program at ORNL and a description of the kinds of investigations that this resource will support.