That depends on the projection used to obtain the map image.

This post is wrong from this point on.

If you draw a three points on the map such that
A_y == B_y && A_x != B_x
A_x == C_x && A_y != C_y

+=====================+
#                     #
#   A-------------B   #
#   |                 #
#   |                 #
#   C                 #
#                     #
+=====================+
[download]

Is A_lat == B_lat && A_long == C_long?

• If so, every pixel represents the same long and lat difference.

  Pixel_lat = (B_lat - A_lat) / (B_x - A_x)
  Pixel_long = (C_long - A_long) / (C_y - A_y)

• If not, the math gets a bit more complicated, but it's still pretty straightforward. You'll probably need a fourth point D such that
  D_x == B_x && D_y == C_y

  +=====================+
  #                     #
  #   A-------------B   #
  #   |             |   #
  #   |             |   #
  #   C-------------D   #
  #                     #
  +=====================+
  [download]

  There surely exists modules to help you here.

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