Yes, Let's Encrypt acknowledges that they perform their validations from multiple observation points on the Internet. They (intentionally) don't provide a list.

This occasionally annoys people who have an idea like "I will firewall everything but whitelist Let's Encrypt" and then find out that while you're welcome to try to puzzle out some way to make this work, Let's Encrypt won't help you and when it breaks you get to keep both halves.

If LetsEncrypt is actually hosted within AWS, then a network request from them to Route53 might not even hit the public internet.