Yes, it will matter for sure, as you certainly can't run dual channel if both modules are mounted in the same channel. But don't worry, the worst thing that could happen is that the system would only run in single channel.
This layout varies between different mobos, but my guess would be that each pair is one channel. Meaning you'd have to populate both slots of the same color.
Wether or not dual channel is enabled, you can see during POSTing on most mobos (you know the memory-count at bootup). If not, use a program like CPU-Z or Sandra to find out for sure.