Hm. Impossible to say given the sparsity of the information. Two thoughts come to mind which may or may not be relevant on your (unstated) platform:

1. On windows, DLLs (which is where your XS code lives) tend to get loaded once per application.

   So, if you have any static data (pointers) that get initialised when the module is loaded, with values that derive from the interpreter, they may not get re-initialised when a new interpreter is loaded.

   I've no idea if this is the case with .so's on other platforms.

2. You mentioned that you get problems when calling methods, but you don't mention (re-)creating the instances on which you are calling those methods.

   If your application code stores any pointers--for example, callback addresses--from the first interpreter and tried to reuse them once you've spawned the second instance, those retained pointers are quite likely to be broken.

Given the generality of the problem description, the best anyone can offer is generalised possibilities for you to look for.