Three teams will be looking to create history this weekend - and four if you include re-formed Macclesfield.
National League South sides Weston-super-Mare and Slough Town and National League Brackley Town have never reached the FA Cup third round before, while the Silkmen, who now play in National League North, did so 11 times in their previous iteration but haven't since they returned in 2020.
Slough welcome Macclesfield on Sunday (12:30 GMT) in one of the ties of the round.
Slough manager Scott Davies said it was an "incredible" opportunity and that his side are full of "confidence, enthusiasm and motivation to go and get a big tie in the next round".
"The FA Cup is special and we're lucky that we're still in it," Davies told BBC Radio Berkshire.
"It's absolutely huge. I think from prize money and being on TV, we've got £126,000 so far. It's obviously a massive bonus for the club but it's well deserved.
"We're the last remaining Berkshire team and it's the first time that any team in Berkshire has stayed in the competition longer than Reading for about 40 years."
The Rebels hold the record for the most second round appearances - a total of nine - without making it to the third round, but Davies knows they face a tough test to break that duck.
"It's not a perfect opportunity because Macclesfield are a really good side but we've certainly got a good opportunity to hopefully get through and you just never know," he added.
"You can then dare to dream of a trip to Old Trafford. I don't want it to be something we regret in years to come. I'm hoping we can put in a performance that gets us through and then we can go into dreamland territory."