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I graduated in 2014, and my situation is that I have been out of school for three years (as of fall 2017 it will be three years). My first recommendation is from an undergraduate research advisor who I've worked with extensively, and who I published with this year, so it's current.

My second is a research advisor from 2015 when I received a fellowship to do to research abroad. He is in a subfield that I want to do my grad research in.

My third is my undergraduate major advisor, who knew me well and who I worked as a TA for for two semesters. We've kept in contact over the years and he's been a real help in helping me receive scholarships/fellowships when he wrote me letters in the past.

My question is this: I had a research experience in the summer of 2014 at a national lab, but I have not asked my advisor there for a letter for most of my schools that only require 3 letters. The research was not in the subfield I'm looking to go into, but it would be the next most recent item on my CV, and more importantly, it would be a research reference. It's not a case of being on bad terms with this person. He is able to write me a strong letter, as I explicitly asked this of him for schools that required a fourth letter (and he easily agreed), but I genuinely think my third letter writer as indicated above is stronger. That professor was instrumental in designing many of the courses in my major department (and so has intimate knowledge of the curriculum and what my grades actually mean in those courses), and is known for writing excellent letters. Additionally, the national lab internship was only for three months, which is a very short amount of time.

I have read on here that omitting such a letter could in some cases be a red flag to admissions committees, especially if it is recent, but I'm wondering if perhaps this case is a bit different. I already have two referees who can speak strongly to my research experience, and they're directly in the subfield I'm interested in. I don't think the other research referee would say anything that these referees have not already touched on, but I do think having the undergraduate advisor, who I've worked for and who has known me for a very long time in an academic context (but non-research context), would be valuable.

What should I do in this case?

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It is not a requirement that you include letters from every research advisor you've ever worked with as part of your graduate application. Since you already have letters from multiple research advisors, you can choose your third letter accordingly. If you believe the letter from your academic advisor is more helpful to you, then you should continue to use that letter. For example, your third advisor could more helpfully comment on your teaching abilities, if that is something you feel is important in your application.

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the short answer to your question is no. The longer answer is that the applications committee will look - holistically at your application including your reference letters. Speaking from the UK context, the recommendation letters do matter but they do not matter as much as you may think. By all means think about whom and how many letters you need but that is not going to prove decisive in your PhD application. Your track record; grades; potential and demonstrated ability for research etc - in my view - matter more.

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