Is it "#1 of a series" or "#1 in a series"?

You might be over-thinking it. That said, I think I prefer the "#1 in a series" more than "#1 of a series". I would be more likely to use "#1 of..." when followed by a specific number, such as: "#1 of 5", whereas I would be less likely to use "#1 in 5".

"#1 in...", using in followed by an abstract quantity/collective sounds better to me, but that may just be my subjective opinion. For example: "#1 in (a) line" compared to "#1 of (a) line" or "#1 in a collection" or "#1 of a collection".

Also, the of gives it the feeling that it is a ranking or score rather than a position in a series.