The next step was to mate each of the G1 males with G1 females derived from crosses of different founders. Their progeny contained DNA from each of 4 founder strains. There is a total of 1,680 nonredundant ways that the G1 mice may be mated. The G2 mice are the parents who will give rise to the family tree of The Collaborative Cross.

By mating the G2 males with G2 females with different combinations of founder DNA, G3 progeny will inherit DNA from all eight of the founders. There are over 40,000 nonredundant ways that the G2 mice may be mated. Each of The Collaborative Cross strains begins by sib-mating the G3 mice.

Different random matings were undertaken at each of the three sites of The Collaborative Cross.