We have examined the pattern of hidden allelic variation at 10 loci in mainland and Bogota populations of Drosophila pseudoobscura by various electrophoretic criteria employing gel concentration, pH, and buffer variation. Based on these loci the genetic similarity between mainland and Bogota populations has decreased from 64 to 15%. Bogota is genetically differentiated at 6 of the 10 loci examined (per locus genetic identity less than 5%). We have also made a comparative study of developmental rate, mating speed, sex ratio, fertility, egg-to-adult viability, and ethological differences and found that for all these fitness characters Bogota is polymorphic and shows some genetic differentiation from the mainland populations of D. pseudoobscura. These results are discussed in relation to the current estimates of genetic similarity between species, and the extent of genetic differentiation during species formation.