I seem to remember (from ages ago) that there was a 300 item limit per folder. I cannot remember if this was an early Twonky 4 limit, or maybe I'm even thinking of something else altogether!
I structured my music into folders called ABC, DEF, GHI etc and put stuff like Aswad, B52s and Cream in the ABC folder etc, etc. Under each artist folder, I then have all the albums (one per folder).
Someone else will maybe chip in and confirm if this limit is true (sorry, I just cannot remember if this was a Twonky issue or something completely different), but if you have a massive collection of folders within a single folder, or maybe even masses of music files in one big folder, maybe this is what's causing the problem. If so, it might be as simple as a bit of house keeping and a database rebuild to sort it all out.
As for TMS5, I'd think it'll be a significant step forward but advise maybe holding on for a week or two; 5.0.XX is not really suited to non technical NAS users (requires Telnet access and some tweaking to make it work) and 5.1 is still really under development. Hopefully the new version of 5.1 will address all the outstanding issues.